Identification of imidazo-pyrrolopyridines as novel and potent JAK1 inhibitors

Article abstract of DOI:10.1021/jm300438j

A therapeutic rationale is proposed for the treatment of inflammatory diseases, such as rheumatoid arthritis (RA), by specific targeting of the JAK1 pathway. Examination of the preferred binding conformation of clinically effective, pan-JAK inhibitor 1 led to identification of a novel, tricyclic hinge binding scaffold 3. Exploration of SAR through a series of cycloamino and cycloalkylamino analogues demonstrated this template to be highly tolerant of substitution, with a predisposition to moderate selectivity for the JAK1 isoform over JAK2. This study culminated in the identification of subnanomolar JAK1 inhibitors such as 22 and 49, having excellent cell potency, good rat pharmacokinetic characteristics, and excellent kinase selectivity. Determination of the binding modes of the series in JAK1 and JAK2 by X-ray crystallography supported the design of analogues to enhance affinity and selectivity.

Full text of DOI:10.1021/jm300438j

Article
pubs.acs.org/jmc
Identification of Imidazo-Pyrrolopyridines as Novel
and Potent JAK1 Inhibitors
Janusz J. Kulagowski,*,† Wade Blair,§,▽ Richard J. Bull,† Christine Chang,§ Gauri Deshmukh,⊥
Hazel J. Dyke,† Charles Eigenbrot,○ Nico Ghilardi,# Paul Gibbons,∥ Trevor K. Harrison,†
Peter R. Hewitt,† Marya Liimatta,§ Christopher A. Hurley,† Adam Johnson,§ Tony Johnson,†
Jane R. Kenny,⊥ Pawan Bir Kohli,§ Robert J. Maxey,† Rohan Mendonca,∥ Kyle Mortara,○
Jeremy Murray,○ Raman Narukulla,† Steven Shia,○ Micah Steffek,○ Savita Ubhayakar,⊥
Mark Ultsch,○ Anne van Abbema,§ Stuart I. Ward,† Bohdan Waszkowycz,‡,⧫ and Mark Zak∥
‡
†Departments of Medicinal Chemistry and Computer Aided Drug Design, Argenta, 8/9 Spire Green Centre,
Harlow CM19 5TR, United Kingdom
∥
⊥
§Departments of Biochemical and Cellular Pharmacology, Discovery Chemistry, Drug Metabolism and Pharmacokinetics,
#Immunology, and ○Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States
S
* Supporting Information
ABSTRACT: A therapeutic rationale is proposed for the treatment of inflam-
matory diseases, such as rheumatoid arthritis (RA), by specific targeting of the
JAK1 pathway. Examination of the preferred binding conformation of clinically
effective, pan-JAK inhibitor 1 led to identification of a novel, tricyclic hinge
binding scaffold 3. Exploration of SAR through a series of cycloamino and
cycloalkylamino analogues demonstrated this template to be highly tolerant of
substitution, with a predisposition to moderate selectivity for the JAK1 isoform
over JAK2. This study culminated in the identification of subnanomolar JAK1
inhibitors such as 22 and 49, having excellent cell potency, good rat pharma-
cokinetic characteristics, and excellent kinase selectivity. Determination of the
binding modes of the series in JAK1 and JAK2 by X-ray crystallography supported the design of analogues to enhance affinity and
selectivity.
the JAK2 gene die of anemia,2a while a gain-of-function muta-
INTRODUCTION
The Janus protein tyrosine kinases (JAK1, JAK2, JAK3, and
■
tion in the JAK2 gene (JAK2V617F) results in polycythemia vera
and other myeloproliferative disorders (MPD) in humans.4
TYK2) mediate intracellular signaling of numerous cytokines
Similar to JAK1 and JAK2, TYK2 is also widely expressed and
and thereby play critical roles in a variety of biological pro-
activated by numerous cytokines. In contrast to JAK1 and
cesses, including hematopoiesis and the regulation of immune
and inflammatory responses.1 JAKs constitutively associate
JAK2, however, deficiency in TYK2 is not lethal, suggesting that
TYK2 might be more dispensable than JAK1 and JAK2. While
with cytoplasmic motifs in the intracellular domain of cytokine
receptors and are activated upon ligand induced receptor
homo- or heterodimerization, which results in the immediate
phosphorylation of tyrosine residues within the cytoplasmic
TYK2-deficient mice show no gross abnormalities in the ab-
sence of challenge, TYK2-deficient humans suffer from hyper
IgE syndrome but are otherwise viable.5 Finally, JAK3 is the
only JAK family member with a restricted expression pattern
domain of the receptors. These phosphotyrosines then serve as
and function, being localized in lymphoid tissue.6 This protein
docking sites for cytoplasmic signal transducer and activator of
transcription (STAT) proteins. Upon recruitment to the recep-
tor complex, STATs become phosphorylated by the JAKs,
associates exclusively with the γc chain, and in both humans and
mice, JAK3 deficiency phenocopies γc deficiency, resulting in
X-linked severe combined immunodeficiency (X-SCID) due to
which allow them to dimerize and travel to the cellular nucleus,
where they act as transcription factors.2
complete abrogation of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21
signaling.7
Biochemical and genetic studies have shown that JAK1 is the
most broadly used JAK, as it is involved in the signaling of the
gamma common (γc), beta common (βc), gp130, type I and
Owing to its restriction to the γc subfamily of cytokines,
JAK3 has long been viewed as an attractive therapeutic target
type II interferon, IL-6, and IL-10 subfamilies of cytokines.1a,3
for the treatment of certain immune system indications (e.g.,
transplant rejection and rheumatoid arthritis (RA)).7,8 Initially
JAK2 utilization is more restricted and generally associated
with receptors that function as homodimers, such as the
erythropoietin (EPO) and growth hormone receptors, among
others. Consistent with its role in EPO signaling, mice lacking
Received: March 29, 2012
Published: May 16, 2012
© 2012 American Chemical Society
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described as a JAK3 specific inhibitor,8b the small molecule
tofacitinib (1, CP-690,550; Figure 1) is now in advanced clinical
Anemia has been observed in some patients during clinical
evaluation of both 121 and 2.22 As noted above, both com-
pounds exhibit potent JAK2 inhibitory activity, and a link be-
tween JAK2 deficiency and anemia has been demonstrated in
mice.22a,23 Therefore, we reasoned that a JAK1 selective, JAK2
sparing inhibitor would constitute an ideal therapeutic agent to
target inflammatory disorders such as RA.24 Herein we describe
the identification and characterization of a novel series of
tricyclic JAK inhibitors along with our initial efforts at achiev-
ing the desired JAK1/JAK2 selectivity profile within this
chemotype.
RESULTS AND DISCUSSION
■
Figure 1. JAK inhibitors in advanced clinical evaluation.
Molecular modeling studies previously performed on com-
pound 1 indicated that a staggered, anti-periplanar conforma-
tion (anti-1, Figure 2), in which the N-methyl group points
toward the pyrrole ring, is energetically favored over the
alternate rotational isomer (syn-1, Figure 2).25 Furthermore, a
single crystal X-ray structure determination of a closely related
derivative indicated this compound also exists in a similar
staggered conformation.26 In contrast, an X-ray cocrystal
structure of 1 bound to JAK1 clearly showed the piperidine
moiety residing in an eclipsed, syn-periplanar orientation to the
bicyclic ring system (syn-1, Figure 2).27 These observations
suggested the use of a tricyclic template to rigidify the inhibitor
structure in a manner that would closely mimic the compound
1 rotamer that was preferred for JAK1 binding. We elected to
explore this possibility by introducing an imidazole ring into the
bicyclic inhibitor design, giving rise to an imidazo-pyrrolopyr-
idine scaffold (e.g., 3, Figure 1).28
At the outset of this work, it was reasoned that, in order to be
considered potential drug candidates, any new compounds
should possess levels of biochemical and functional JAK1
potency comparable to 1. Specifically, the goal was to achieve at
least low single-digit and, preferably, subnanomolar biochemical
activity, with a cellular EC50 value ideally less than 100 nM.
However, the degree of selectivity for JAK1 over JAK2 that
would be required to impart therapeutic benefit relative to 1
was unclear. The observation of reduced hemoglobin levels at
higher (>5 mg BID) doses of 1 during a recent clinical trial was
attributed by the authors to possible effects on hematopoiesis
through inhibition of JAK2.21b This led us to surmise that a
relatively modest degree of selectivity for JAK1 over JAK2,
perhaps on the order of 10-fold, may be sufficient to provide a
suitable therapeutic window.
testing and has demonstrated efficacy against RA, psoriasis,
Crohn’s disease, kidney transplant rejection, and ulcerative
colitis.9 It has recently become clear that 1 is not as specific as
initially thought for JAK3, but rather is a potent pan-JAK
inhibitor (Ki = 0.68, 0.99, 0.24, and 4.39 nM for JAK1, JAK2,
JAK3, and TYK2, respectively).10 Multiple inhibitors targeting
JAK2 are also undergoing clinical evaluation against MPDs, and
several have exhibited promising results.4,11 Of these agents,
ruxolitinib (2, INCB018424; Figure 1) was approved in 2011
for the treatment of myelofibrosis,12 and the compound has
also demonstrated efficacy in the RA setting.13 The reported
potency of ruxolitinib (2) for JAK3 is far weaker than its
inhibitory activity toward the other JAK family members
(IC50 = 3.3, 2.8, 428, and 19 nM for JAK1, JAK2, JAK3, and TYK2,
respectively),14 suggesting that the compound’s clinical efficacy
against RA is unlikely to be driven by inhibition of the JAK3
isoform. Furthermore, a recent report claims that for cytokine
signaling mediated through γc-containing receptors, the JAK1
pathway is dominant over JAK3.15
Both 1 and 2 are potent inhibitors of JAK1 and JAK2, but
differ in their potency for JAK3 and TYK2. Since both have
exhibited activity against RA, we hypothesized that this
property was a result of JAK1 inhibition as a result of the
following considerations. First, while IL-6 stimulation activates
JAK1, JAK2, and TYK2, cells taken from JAK1 knockout mice
exhibit substantially reduced levels of responsiveness when
compared to those from wild-type animals.16 In contrast,
comparable activities toward IL-6 are observed between wild-
type and knockout mice in the case of both JAK217 and
TYK2.18 Second, tocilizumab, an IL-6R-specific, neutralizing
humanized monoclonal antibody, has shown impressive clinical
efficacy in the treatment of RA.19 Furthermore, JAK1 rather
than JAK2 participates in the signaling of pathophysiologically
relevant γc cytokines, including IL-15, neutralization of which
also results in improvement of RA.20
To the best of our knowledge, no account of how JAK1
selectivity may be achieved has thus far been disclosed,
although compounds with this profile have been described in
recent patent applications.29 Moreover, a compound (GLPG0634;
structure undisclosed), reported to be JAK1 selective (IC50 = 10, 28,
810, and 116 nM for JAK1, JAK2, JAK3, and TYK2, respectively),30
Figure 2. Generation of tricyclic inhibitor scaffold 3.
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Table 1. JAK Isoform Inhibitory Activities of Heterocycloamine Derivatives of 3
a
b
Arithmetic mean of at least 3 separate determinations (n ≥ 3).34 Not applicable.
has recently demonstrated efficacy against RA with a good
safety profile in an early phase II trial.31 Furthermore, diamino-
1,2,4-triazoles structurally related to JNJ-7706621 (IC50 = 39,
1700, 950, and 39 nM for JAK1, JAK2, JAK3, and TYK2,
respectively) have been reported to also be selective for JAK1
and TYK2 over the other JAK isoforms,32 although the refer-
ence compound is known to be somewhat promiscuous against
a variety of other kinases.33
Figure 3. Synthetic precursor to 1.
In our biochemical assays,34 compound 1 was confirmed to
be a potent, pan-JAK inhibitor with some selectivity over TYK2
(Table 1). Gratifyingly, the racemic tricyclic molecule 4
displayed similar inhibitory properties when tested against the
same enzymes. Equally striking was the observation that, while
5 (Figure 3), an N-benzyl-containing compound related to 1,
was several hundred-fold less potent, the corresponding tri-
cyclic inhibitor 6 suffered only an order of magnitude loss in
activity. These observations suggested that the tricyclic imidazo-
pyrrolopyridine scaffold could possess a divergent and productive
SAR when compared to pyrrolopyrimidine-containing inhibitors
such as 1. Simplification of 4 by the removal of the piperidinyl
methyl group afforded 7, which maintained excellent JAK1
potency.
Encouraged by these preliminary results, we sought
alternatives to the distinctive cyanoacetamide substituent
present in 1, 4, and 7. Capitalizing on the favorable results
observed for 6, we examined different ring systems that retained
the N-benzyl group (Table 1). The subtle contribution to JAK
binding of the piperidinyl methyl group present in 6 was
demonstrated by the des-methyl analogue 8 (2−6-fold loss in
potency across the four isoforms), while 9 confirmed that the
(R)-piperidine configuration favored JAK1 activity. Replace-
ment of the piperidine in 8 with the corresponding N-benzyl
pyrrolidine and azetidine had minimal impact on inhibitory
potency across the four JAK family enzymes in our testing
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panel (10 and 11, respectively). However, incorporation of an
achiral 4-substituted piperidine into the tricyclic inhibitor design
(12) resulted in a dramatic increase in JAK potency relative to
the 3-substituted piperidines 8 and 9.
of properties, with excellent cell potency and high in vitro
metabolic stability.
Some general observations on the tricyclic compounds
discussed thus far warrant comment. 4-Piperidine tolerated a
wide range of subtituents; the varied chemotypes contained in
Table 2 span only a 100-fold range in JAK1 inhibitory activity.
The tricyclic scaffold also appeared to have an intrinsic
preference for JAK1; nearly all of the compounds displayed a
very modest but consistent selectivity for this isoform over
JAK2 in the biochemical assays. Furthermore, excellent
JAK1-pSTAT3 cell potency was achievable with the tricyclic
inhibitors. Cell and biochemical selectivities were in good
agreement for the most part, with perhaps a subtle enhance-
ment of the former relative to the latter, as observed also with 1.
Derivatization of the piperidine with dimethyl glycine (23)
resulted in some loss in enzyme activity, but an improvement in
biochemical selectivity, when compared to the isosteric amide
(17). This characteristic was observed with other derivatives
bearing a basic center in this vicinity, with cyclization (24−27)
generally offering some benefit to both biochemical potency
and selectivity. For instance, proline derivative 26 displayed a
17-fold preference for JAK1 over JAK2. Possible reasons for the
enhanced selectivity observed with compounds incorporating a
basic, pendent substituent are discussed in more detail below.
Unfortunately, despite possessing intriguing levels of selectivity,
progression of this subset of compounds was precluded by a
persistent inability to achieve required levels of inhibitory
activity, leading to commensurately poor cellular potency.
With the aim of enhancing binding interactions by exploiting
potential lipophilic contacts between the piperidine ring and
protein residues located under the P-loop (see discussion of
crystallographic studies below), each position on the ring was
sequentially probed with a methyl group. Incorporation of a
methyl group in both cis and trans dispositions at either the
2- or 3-positions on the piperidine ring provided no significant
benefit (data not shown); however, a distinct trend emerged
when this substituent was located at the 4-position (Table 3).
Although this change generally reduced JAK1 enzyme potency
relative to the corresponding unmethylated counterparts, the
extent was dramatically dependent on N-substitution. Thus,
sulfonamide 30 was equipotent to its unmethylated analogue
19, whereas 32 suffered a 50-fold reduction in activity when
compared to 22. However, in each case, biochemical selectivity
over JAK2 was improved relative to the unsubstituted example,
although this effect was variable, ranging from 3-fold for 32 to
16-fold for 28. The general reduction in activity observed on
methylation of the piperidine also translated to the cellular
assay; of the compounds tested, only 30 displayed moderate
potency.
Having identified compounds such as 7 and 12 with en-
couraging JAK inhibitory potency, further optimization of this
novel series of tricyclic JAK inhibitors was undertaken. Follow-
ing the determination of enzyme activity against all four
isoforms, compounds were assessed for cellular potency
and selectivity. Since the general pattern of isoform activity
displayed in Table 1, namely higher inhibition at JAK1 and
JAK2 than at JAK3 and TYK2, was maintained in subsequent
examples, further discussion of biochemical potency against the
latter two enzymes will be omitted for the purpose of brevity.
JAK1 enzyme (biochemical) selectivity is expressed as the ratio:
JAK2 (Ki)/JAK1 (Ki). With regard to cellular potency, it was
considered desirable to determine both JAK1 and JAK2 cellular
activity in the same cell line, in order to circumvent any issues
arising from differences in response between alternate cell
types. In TF-1 cells, IL-6 stimulation leads to STAT3 phospho-
rylation predominantly through involvement of JAK1, whereas
EPO stimulation elaborates phospho-STAT5 in a JAK2
dependent fashion.34 By analogy to enzyme selectivity, quoted
cell selectivities are the ratios of pSTAT5 EC50 over pSTAT3
EC50. Compounds were also routinely evaluated for metabolic
stability by incubation with human liver microsomes. Reference
standard 1 exhibited excellent cell potency in the IL-6-driven
pSTAT3 cell assay (Table 2). Despite having comparable JAK1
biochemical potency, tricyclic analogue 7 exhibited somewhat
weaker cell potency, but equivalent microsomal stability to 1.
Although having somewhat reduced activity for JAK1 relative to
7, 12 proved more potent in the JAK1-pSTAT3 cellular assay.
However, in vitro turnover in human microsomes was observed
to be higher for 12 than that noted for 7. Despite this short-
coming, the 4-piperidinyl regioisomer was selected for further
optimization, on the basis that a cyanoacetyl substituent was
not required, as demonstrated by the excellent JAK1 enzyme
potency of 12, and the fact that this positional isomer was less
stereochemically complex by virtue of not possessing a chiral
center. Reasoning that the high in vitro clearance observed with
12 was due to the presence of a lipophilic benzyl group, efforts
were undertaken to address this liability. Unsubstituted pipe-
ridine 13 suffered a significant loss in JAK1 inhibitory activity,
with a concomitant loss in cellular potency. Introduction of
polarity into 12 in the form of a pyrimidine (14) maintained
comparable inhibitory activity, while concurrently reducing de-
gradation in human microsomal preparations. These improve-
ments unfortunately came at the expense of cellular potency
when compared to the less polar 12. The cyanoacyl substituent
present in 1, 4, and 7 was significantly less successful when
introduced in the context of the 4-piperidine (15), especially in
terms of cellular activity. A benzoyl 4-piperidine substituent
likewise proved to be a poor replacement for benzyl (16). No
JAK1 potency advantage was offered by a simple alkyl amide
(17), although JAK1 enzyme potency was maintained relative
to 12 and 14 with sulfonamide 19 and, to a lesser extent, 18.
Interestingly, substitution of the 4-piperidine with a simple alkyl
group, as in 20, was highly detrimental to activity. However,
functionalization of the alkyl group with electron-withdrawing
moieties proved to be a particularly successful strategy, with
both JAK1 enzyme and cell potency being restored in the
trifluoroethyl analogue 21. Within this series of compounds,
cyanoethyl derivative 22 encompassed the optimum overall balance
In an effort to improve further upon the properties of the
compounds described thus far, a series of substituted cyclo-
hexylamines was also explored. For structural simplicity, and by
analogy with the findings from an evaluation of piperidine SAR,
attention in this case was focused on the 1,4 disubstituted
regioisomer. Additionally, a selection of cis and trans stereo-
isomeric pairs was examined in order to establish stereo-
chemical preference.
Although identical JAK1 inhibitory activity was exhibited by
tert-butyl carbamates 33 and 34 (Table 4), a marginal prefer-
ence for the trans over cis isomer was observed in the case of
the unsubstituted amine (35 and 36, respectively). However,
this trend was far more pronounced in the morpholine sub-
stituted pair, with the trans isomer 37 significantly more potent
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Table 2. In Vitro Properties of 4-Piperidinyl Derivatives of 3
a
b
c
Arithmetic mean of at least 3 separate determinations (n ≥ 3). Biochemical selectivity for JAK1 over JAK2 (JAK2 Ki/JAK1 Ki). pSTAT3-IL6
d
e
JAK1 driven TF-1 cell-based assay. Cell-based selectivity for JAK1 over JAK2 (pSTAT5-EPO EC50/pSTAT3-IL6 EC50). Half-life in the presence of
in vitro preparations of human liver microsomes. From ref 35. Not determined. Single, more active enantiomer; absolute configuration was
f
g
h
assigned arbitrarily.
than the cis isomer 38. The trans diastereomer also possessed
superior cell potency in the case of the t-butyl carbamate
(33, 34) and morpholine (37, 38) pairs, although not for
amines 35 and 36. Indeed, both latter compounds displayed
very similar profiles, having moderate biochemical and cell
potencies, but high (12-fold) JAK1 cellular selectivites.
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Table 3. In Vitro Properties of 4-Methylated Piperidinyl Derivatives of 3
a
b
c
Mean of at least 3 separate determinations (n ≥ 3). Biochemical selectivity for JAK1 over JAK2 (JAK2 Ki/JAK1 Ki). pSTAT3-IL6 JAK1 driven
d
e
TF-1 cell-based assay. Cell-based selectivity for JAK1 over JAK2 (pSTAT5-EPO EC50/pSTAT3-IL6 EC50). Half-life in the presence of in vitro
preparations of human liver microsomes.
However, further evaluation of 35 indicated that this com-
pound had poor rat pharmacokinetic characteristics (see below).
Derivatization of 35 provided a range of substituents broadly
comparable to those applied to the piperidines in Table 2.
Similar tolerance of a broad range of substitution was also
observed with the examples listed in Table 4. Amide 39, amines
40−42, and sulphonamide 43 all possessed single or low
double digit nanomolar potency for JAK1 but with variable
cellular selectivities. Although having a compelling pharmaco-
logical profile, carbamate 33 displayed only moderate micro-
somal stability. Reasoning 33 was compromised by its high
lipophilicity (logD7.2 3.2), we sought to address this issue by
preparing the corresponding methyl analogue 44 (logD7.2 1.9).
While this change dramatically improved metabolic stability, it
proved detrimental to both enzyme and cell potency and
selectivity, relative to 33. Interestingly, N-methylation of the
carbamate (45) had minimal impact on potency but appeared
to abolish both biochemical and cell selectivity. Seeking to reduce
hydrophobicity further, the cycloalkyl moiety was truncated to a
cyclopentyl ring. Racemic trans derivative 46 (logD7.2 1.2)
displayed a 3-fold improvement in JAK1 biochemical activity
over 44, with a similar enhancement in cell potency. Further
improvement in cell potency and selectivity was realized on
reinstatement of a t-butyl carbamate (47) but with no penalty in
metabolic stability relative to 46. The corresponding cis analogue
48 was less successful, both in terms of potency and stability.
Resolution of 47 provided 49 as the more active enantiomer.
With subnanomolar enzyme activity against JAK1, this com-
pound also proved to have excellent potency in the pSTAT3 cell
assay, with an overall profile comparable to that of 1. However,
in contrast to the potent but unselective 1, compound 49
displayed moderate selectivity for the JAK1 isoform over JAK2,
both in biochemical and cell-based assays, in addition to a
superior microsomal stability profile.
since the volume of distribution for most examples was moderate,
save for compounds 12 and 35, for which the much higher
values were presumably a reflection of their overtly basic nature.
Despite high clearance, reasonable plasma exposure was
achieved following PO dose for some compounds, depending
on the balance of properties. A similar profile was previously
reported for 1.26 The poor oral exposure of 7 was in keeping
with low MDCK apparent permeability, which likely resulted
from a combination of low lipophilicity (logD) and high
polarity (TPSA). In contrast, N-benzyl piperidine 12 exhibited
excellent permeability, but its high lipophilicity and low polarity
resulted in high in vitro turnover, with correspondingly high
in vivo clearance, culminating in a very poor rat PK profile. These
results suggested that an appropriate balance between logD and
TPSA was required in order to achieve an appropriate level of
permeability with perhaps some modulation of clearance in
support of a favorable PK profile. Compounds 19, 30, and 41
possessed near identical logD and TPSA values (intermediate
between those of 7 and 12) and consequently displayed similar
permeability. The PK characteristics of all three compounds
showed a substantial improvement over those of 7 and 12, with
19 and 30 in particular having good exposure and oral bio-
availabilty. Although having comparable and moderate TPSA,
22 and 35 exhibited widely disparate PK parameters. The
higher lipophilicity of 22 conveyed an excellent overall profile,
whereas that of 35 was compromised by its remarkably low
logD and poor apparent permeability, despite having lower
clearance. Likewise, although both 12 and 49 are very lipo-
philic, this potential liability is offset to some extent by the
significantly higher polarity of the latter, resulting in 49 having
very favorable PK properties.
To assess pan-kinase selectivity, compounds were screened at
a concentration 100-fold higher than their JAK1 Ki against
a commercial panel of 50 (183 in the case of 7) kinases.
Compounds were scored by the number of proteins showing
>50% inhibition. On this basis, 7, 22, and 49 proved to have
excellent selectivity against non-JAK kinases, while 35 and 41
were less selective.
Selected compounds were subjected to more stringent
evaluation, including pharmacokinetic profiling in rat and
determination of off-target selectivity (Table 5). In vivo plasma
clearance following IV dose was high for all compounds. This
was unlikely to be as a result of extensive tissue distribution
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Table 4. In Vitro Properties of Cycloalkylamine Derivatives of 3
a
b
c
Relative stereochemistry of substituents on the cyclohexane or cyclopentane ring. Mean of at least 3 separate determinations (n ≥ 3). Biochemical
d
e
selectivity for JAK1 over JAK2 (JAK2 Ki/JAK1 Ki). pSTAT3-IL6 JAK1 driven TF-1 cell-based assay. Cell-based selectivity for JAK1 over JAK2
f
g
h
(pSTAT5-EPO EC50/pSTAT3-IL6 EC50). Half-life in the presence of in vitro preparations of human liver microsomes. Not determined. Racemic.
i
Single, more active enantiomer; absolute configuration was assigned arbitrarily.
acids in close contact to the ligand are conserved or show
modest changes (e.g., JAK1 Phe958 → JAK2 Tyr931 on the
hinge region). More significant differences arise in solvent-
exposed residues at the periphery of the binding site, including
Arg879 → Gln853, Glu966 → Asp939, and Lys965 → Arg938.
Although there are several changes to P-loop residues (e.g.,
Glu883 → Lys857, His885 → Asn859), the side chains point
away from the ligand and hence are not likely to be amenable
for the design of direct hydrogen bonding contacts. The high
X-RAY CRYSTALLOGRAPHIC STUDIES
■
X-ray crystallography proved useful in establishing the binding
mode of various members of the imidazo-pyrrolopyridine series
and interpreting SAR trends across the JAK isoforms. The
X-ray structures of 1 bound to all four JAK isoforms have been
reported in the literature.27,36 Consistent with its pan-JAK
activity, 1 exhibits a similar binding mode to all four JAK
isoforms. A high degree of sequence homology exists between
JAK1 and JAK2 within the ATP-binding site (Figure 4). Amino
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Table 5. Physicochemical, in Vitro, and Rat Pharmacokinetic Properties of Selected Compounds
d
in vivo rat PK
TPSA
(Å2)
MDCK A:B
rat Hep.
CL
AUC
(μM·h)
C
Vss
(L/kg)
F
(%)
kinase select.
e
a
b
c
f
g
maxh
i
j
Ex logD
Papp (×10−6cm/s)
T1/2 (h)
(mL/min/kg)
(μM)
(>50% inhib.)
k
1
88
90
49
83
73
83
72
82
84
62
0.7
27
5
7
0.39
0.3
>10
0.5
85
0.3
0.05
0.04
2.9
1.87
6.39
1.46
2.37
2.72
13.9
3.96
2.53
2/183
1/50
1/50
0/50
3/50
8/50
5/50
0/50
l
12 2.90
19 1.04
22 1.72
30 1.14
32.3
1.2
170
45
BLQ
<1
36
48
74
<1
28
73
>10
>10
>10
>10
9.8
2.3
1.6
2.3
0.22
0.7
1.3
8.2
96
1.8
2.5
82
0.8
35 −0.44
41 0.97
49 2.99
0.2
65
0.02
0.6
1.5
120
140
11.2
2.2
1.1
a
b
c
Measured at pH 7.2. Apparent permeability in MDCK transwell culture, A:B apical to basolateral. In vitro stability in cryopreserved rat
d
e
hepatocytes. Normalized to 5 mg/kg po, 1 mg/kg iv. Number of enzymes inhibited >50% out of a panel of 183 or 50 non-JAK kinases, at a test
f
g
h
concentration of 100 × JAK1 Ki. Plasma clearance following iv dosing. Plasma area under the curve following po dosing. Maximal plasma
concentration following po dosing. Volume of distribution at steady state. Oral bioavailability. Data taken from ref 26. Below the level of
i
j
k
l
quantification.
Figure 4. Comparison of the X-ray structures of 1 complexed with
JAK1 (white carbon atoms; PDB ID 3EYG27) and JAK2 (cyan carbon
atoms; PDB ID 3FUP), showing the conserved binding mode of the
ligand and the overall similarity in tertiary structure of the JAK
isoforms. The phosphate-binding loop (P-loop), activation loop (A-
loop), and hinge region are labeled. Residue differences between JAK1
and JAK2 that are located close to the ATP-binding site are shown in
thin stick format. Residue numbers refer to JAK1. Figure was
generated with Pymol.38
Figure 5. Comparison of the X-ray structures of 1 complexed with
JAK1 (white carbon atoms; PDB ID 3EYG27) and 7 complexed with a
JAK2 triple mutant (green carbon atoms) showing the conserved
binding mode. The phosphate-binding loop (P-loop), activation loop
(A-loop), and hinge region are labeled. Residue numbers refer to
JAK1. Figure generated with Pymol.38
peptide backbone of the P-loop (e.g., the carbonyl carbon of
Gly887) and the catalytic Lys908. The tight contacts of this
group under the P-loop may explain the loss of potency for
bulkier groups in this position on the 3-piperidine core (e.g., 8).
Transfer of the cyanoacetate to the 4-piperidine core (15)
results in a loss of activity: molecular modeling studies suggest
the acetonitrile group of 15 points along a different vector
compared to 7 and hence is unable to bind as tightly under the
P-loop.
In general, crystallographic studies revealed that small
N-substituents on the 4-piperidinyl core sit loosely between the
P-loop and the C-terminal lobe, close to residues associated
with the binding of the phosphate of ATP, such as Asn1008 and
degree of homology suggested that improvement of selectivity
for JAK1 over JAK2 would not be a trivial task.37
As desired in the original design, the binding mode of 1 is
conserved in tricyclic analogue 7 (Figure 5). The pyrrolopyr-
idine ring system of 7 binds to the hinge region (JAK1 resi-
dues Glu957 and Leu959) in a way similar to that of the
pyrrolopyrimidine core of 1. The piperidine ring binds to a
small lipophilic pocket adjacent to Leu1010 (on the C-terminal
wall), while the cyanoacetate group is directed deep under the
P-loop. The nitrile does not form any direct hydrogen bonds
but may make favorable electrostatic interactions with the
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Asp1021 in JAK1. Larger substituents on the 4-piperidinyl, such
as the benzyl group of 12, were observed to be oriented to-
ward the P-loop but caused some displacement of the P-loop
compared with 1 and 7 (Figure 6). The observation that
N-Substitution of the 4-piperidine core with acetylamino
groups (23−27) results in a general decrease in inhibitory
activity for JAK1 but a gain in selectivity over JAK2. These
groups are observed crystallographically to extend toward the
bulk solvent between the edge of the P-loop and the C-terminal
lobe. The SAR of these basic substituents may be modulated by
electrostatic interactions with neighboring residues on the
surface of the protein. The presence of several basic amino
acids in this region on the C-terminal lobe (e.g., JAK1 Lys965,
Arg1007) may impair the JAK1 potency of 23−27 due to
electrostatic repulsion: the guanidine group of Arg1007 is
located approximately 5 Å from the pyrrolidine nitrogen of 26.
Computational modeling studies (described in the Supporting
Information) were used to compare the binding of 26 to JAK1
and several single residue mutants of JAK1 mimicking the
corresponding residue changes in JAK2. These calculations
suggested that the residue difference between Glu883 (JAK1)
and Lys857 (JAK2) may drive the selectivity of 26 and similar
molecules for JAK1 over JAK2. A visual comparison of the vacuum
electrostatic potential of JAK1 to that of the in silico JAK1-E883K
mutant illustrates this distinction: the increased positive electro-
static potential in the mutant is repulsive toward the positively
charged pyrrolidine moiety on 26 (Figure 7). This is also
consistent with the observation that other basic analogues, similar
to 26, are disfavored to a greater extent in JAK2 than in JAK1.
Methylation at the 4-position of the 4-piperidinyl group
(Table 3) may have an effect on the SAR either by a direct
interaction with the protein or by stabilization of the ligand
conformation. The piperidinyl group is capable of adopting
several low energy conformations with respect to the tricyclic
core, due to rotation around the bond between the piperidine
and the imidazo ring of the tricycle. In X-ray structures of
3-piperidinyl analogues, including 1, the hydrogen atom at the
3-position of the piperdinyl moiety (i.e., that on the carbon atom
at the point of attachment of the piperidinyl ring to the tricycle)
is directed toward the pyrrolo ring of the hinge binder; this
conformation allows the N-substituent to be directed under the
Figure 6. Comparison of the X-ray structures of 7 (green carbon
atoms) and 12 (orange carbon atoms), both complexed with a JAK2
triple mutant, highlighting movement of the P-loop to accommodate
the benzyl substituent of 12. Residue labels refer to JAK1 numbering.
N-substituents on the 4-piperidine are bound loosely along the
relatively spacious cleft between the P-loop and the C-terminal
lobe may explain why a fairly wide range of substituents is
tolerated at this position (Table 2).
Figure 7. Electrostatic potential representations for the X-ray structure of 26 bound to JAK1 (left) and an in silico model of 26 bound to a JAK1-
E883K mutant (right). Ligands are displayed in stick format with green carbon atoms. Surfaces colored by electrostatic potential from red (negative)
to blue (positive). A similar orientation of each protein is shown. Surfaces and images were generated with Pymol’s protein contact potential
algorithm which approximates the potential that would be felt by a point-charge one solvent radius above the protein surface (Schrodinger, Inc.).
̈
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P-loop. In the 4-piperidinyl series, a broader range of piperidine
rotamers is observed crystallographically, presumably influ-
enced to some extent by the binding interactions of sub-
stituents on the piperidinyl ring. Thus, methylation on the
4-position may serve to stabilize or destabilize specific con-
formations of the piperidine (e.g., due to steric interactions with
the pyrrolo ring of the hinge binder) and thereby influence the
binding interactions of substituents on the piperidinyl ring, as
well as modulate direct lipophilic contacts between the pipe-
ridinyl ring and the C-terminal wall (e.g., Leu1010) (Figure 8).
Figure 9. Comparison of the X-ray structures of 30 bound to JAK1
(white carbon atoms) and JAK2 (cyan carbon atoms), highlighting a
shift in the binding mode and significant movement of the P-loop.
Figure 8. Comparison of the X-ray structures of 12 in JAK2 triple
mutant (orange carbon atoms) and 30 in JAK1 (white carbon atoms),
highlighting different piperidine rotamers due to 4-methylation in 30.
X-ray structures also revealed that the piperidinyl ring of 30
adopted different rotamers when bound in JAK1 and JAK2; the
latter was associated with significant movement of the P-loop
into the binding site in order to maximize contacts with the
inhibitor (Figure 9). This difference in protein conformation
may be an experimental artifact, possibly due to crystal packing
contacts which were present in JAK2 but absent in JAK1; it was
also noted that the electron density for the P-loop was less
complete for the JAK1 complex. The significant shift in the
ligand binding mode might have been expected to result in a
much greater difference in potency between JAK1 and JAK2,
and was not observed for similar inhibitors. However, it is also
worth noting that there are several residue differences between
JAK1 and JAK2 along the P-loop; although these side chains are
directed away from the ligand-binding site, it is possible that they
may affect the flexibility of the P-loop such that different low-energy
conformations may be accessed by JAK1 compared with JAK2.
In general, compounds in the cyclohexyl and cyclopentyl series
(Table 4) were observed to bind similarly to the piperidinyl series.
As may be expected, substituents on the cyclopentyl ring are
directed along a vector intermediate between the vectors presented
by the 3-piperidinyl and the 4-piperidinyl rings, and hence may
show somewhat different trends in SAR. For example, the relatively
bulky carbamate substituent of 49 lies along the groove between
Figure 10. Comparison of the X-ray structures of 7 bound to JAK2 triple
mutant (green carbon atoms) and 49 bound to JAK1 (white carbon
atoms) to demonstrate the different vectors adopted by the substituents on
the 3-piperidinyl and cyclopentyl rings, respectively. A potential hydrogen
bond is highlighted between 49 and D1021 (yellow dotted line).
the P-loop and the C-terminal wall, forming a single direct
hydrogen bond to the protein to Asp1021 (Figure 10).
CHEMISTRY
■
Protection of 4-chloro-7-azaindole 5039 with benzenesulfonyl
chloride provided 51 (Scheme 1), which was regioselectively
nitrated at the 5-position using tetrabutylammonium nitrate in
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a
Scheme 1. Preparation of Heterocycloamine Derivatives of 3
a
−
Reagents and conditions: (i) PhSO2Cl, DMAP (cat.), NEt3, DCM, rt, overnight, 83%; (ii) n-Bu4N+NO3 , TFAA, DCM, −5 °C-rt, overnight, 79%;
(iii) R′NH2, EtN(i-Pr)2, i-PrOH, 120 °C in microwave, 11−20 min, 80−100%; (iv) Fe, NH4Cl, EtOH, H2O, reflux, 70−99%; (v) (EtO)3CH,
p-TsOH (cat.), PhMe, reflux, 18 h, 81−100%; (vi) NaOH, MeOH, H2O, rt, 7 h, 72−98%; (vii) Pd(OH)2 on C, NH4HCO2, MeOH, reflux; (viii)
NCCH2CO2H, HOBT, DMAP, EDCl, DCM, rt, 24 h, 11−48% 2 steps; (ix) Pd on C, H2, THF; (x) TFA, DCM, 94%, then PhCHO, Na(OAc)3BH,
AcOH, DCE, MeOH, rt, 91%.
a
the presence of trifluoroacetic anhydride.40 SNAr displacement
Scheme 2. Preparation of 4-Piperidinyl Analogues
of chloride in key intermediate 52 with amines 53a−f afforded
54a−f. Amine 53a was prepared according to the published
procedure.41 Iron-mediated reduction42 followed by cycliza-
tion proceeded uneventfully, and the product was deprotected
under basic conditions to give the compounds shown in
Table 1 (6, 8−10, and 12). In the case of azetidine 11, the
precursor t-butyl carbamate was deprotected using TFA and
the resulting secondary amine reductively alkylated with
benzaldehyde. Transfer hydrogenolysis of the N-benzyl groups
of 6 and 8, with subsequent cyanoacetylation provided 4 and 7,
respectively.
Hydrogenolysis of 12 provided 13, which was derivatized
under standard conditions (Scheme 2) to give the compounds
in Table 2.
Piperidine derivatives bearing a 4-methyl group were pre-
pared in accordance with the sequence depicted in Scheme 3.
Curtius rearrangement of commercially available Boc-protected
piperidine carboxylate 55 in the presence of benzyl alcohol
afforded bis carbamate 56. Selective deprotection gave amine
57, which required significantly more forcing conditions than
those employed previously to react with 52 on account of its
hindered nature. Manipulation of the adduct in a manner
analogous to that described above gave 58, which on depro-
tection furnished 28. Derivatization of the piperidine provided
the other compounds listed in Table 3.
a
Reagents and conditions: (i) Pd(OH)2 on C, NH4HCO2, MeOH,
reflux, 52%; (ii) Na(OAc)3BH, AcOH, DCE, MeOH, rt, 57−68%; (iii)
Synthesis of cyclohexylamine derivatives 33, 35, and 39−45
15, 16, X = OH HOBT, DMAP, EDCl, DCM, rt, 32−57%; 17, X = Cl
followed an analogous process (Scheme 4), starting with
NEt3, DCM, rt, 52%; 23, 25−27, X = OH HATU, EtN(i-Pr)2, DMF;
monoprotected trans-cyclohexyl-1,4-diamine 59. Compounds
34 and 36 were prepared in a fashion corresponding to that of
33 and 35, respectively, only using the cis diastereomer of 59.
24, then TFA, DCM, then HCHO, Na(OAc)3BH, AcOH, DCE; (iv)
EtN(i-Pr)2, DMF, THF, 13−18%; (v) NEt3, DCM, DMF, rt, 46%; (vi)
EtOH, reflux, 87%.
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a
Scheme 3. Preparation of 4-Methyl Piperidinyl Analogues
a
Reagents and conditions: (i) Ph2P(O)N3, NEt3, PhCH2OH, PhMe, reflux; (ii) Pd on C, H2, EtOH, 47% 2 steps; (iii) 52, EtN(i-Pr)2, i-PrOH,
reflux, 65 h, 79%; (iv) Fe, NH4Cl, MeOH, H2O, reflux, 84%; (v) (EtO)3CH, p-TsOH (cat.), PhMe, reflux, 86%; (vi) NaOH, MeOH, H2O, rt, 98%;
(vii) TFA, DCM, rt, 98%; (viii) pyrimidine-5-carboxaldehyde, Na(OAc)3BH, AcOH, mol. sieves, DCE, rt, 47%; (ix) MeSO2Cl, NEt3, DCM, rt, 58%;
(x) TfOCH2CF3, NEt3, DCM, DMF, rt, 87%; (xi) acrylonitrile, EtOH, reflux, 57%.
Morpholine derivatives 37 and 38 were prepared (Scheme 5)
by reductive amination of cyclohexanone 65 with morpholine,
followed by separation of diastereomers 66 and subsequent
deprotection. Compound 65 arose through oxidation of alcohol
64, itself obtained through a process analogous to that described
previously, starting with amino alcohol 63,
The preparation of cyclopentylamine derivatives 47−49 is
described in Scheme 6. Palladium-mediated reduction of
bicyclic hydroxylamine 6743 followed by N−O bond cleavage
with Raney nickel44 afforded protected cis amino alcohol 68.
Mesylation, azide displacement45 and subsequent reduction
gave trans diamine derivative 70. This was progressed as pre-
viously described to sulfonyl-protected tricycle 72, which gave
47 on basic hydrolysis. This was subjected to SFC resolution,
giving 49 as the more active enantiomer. Conversely, 73 was
prepared by sequential reaction of 72 with TFA and methyl
chloroformate, followed by hydrolysis as before to 46. The cis-
diastereomer 48 was synthesized in a manner analogous to that
for 47, following Mitsunobu inversion of 68 to 69.46
assays, respectively. Moreover, good agreement was generally
observed between the selectivity in both biochemical and
cellular assays for any given compound. A further feature of this
series was the excellent JAK1 inhibitory activity displayed by
the majority of examples. The accommodation of a wide variety
of chemotypes allowed for ready modulation of physicochem-
ical properties, thereby providing the means for achieving good
correlation between biochemical and cell potencies, and
favorable rat pharmacokinetic characteristics.
X-ray crystallography proved valuable for confirming the
general binding mode of the series and also demonstrated the
subtle differences in binding contacts achievable by changes to
substitution patterns. In particular, structural information was
useful in guiding the design of analogues toward regions of the
protein that may be expected to yield improvements in affinity
and selectivity. Further optimization of this series, particularly
with regard to improving selectivity and demonstrating in vivo
activity in a relevant model of inflammatory disease, will be
disclosed elsewhere.
CONCLUSIONS
EXPERIMENTAL SECTION
■
■
Taking account of reported clinical experience with pan-JAK
inhibitors and the important role of IL-6 in mediating inflam-
matory response, it was postulated that selective modulation of
the JAK1 signaling pathway represented a valid strategy for the
potential treatment of pro-inflammatory conditions such as RA.
A significant, recent increase in patent applications claiming
kinase inhibitors with selectivity for the JAK1 isoform would
suggest this to be an emerging area of interest.
General Methods. Commercial reagents and solvents were used
as supplied. Use of ethanol as reaction solvent refers to industrial
methylated spirit. Moisture or oxygen sensitive reactions were con-
ducted under an atmosphere of argon or nitrogen gas. Chemical and
optical (where applicable) purities were >95% for all final compounds
as assessed by quantitative HPLC with DAD UV detection and chiral
SFC analysis, respectively. Further details on the analytical con-
ditions used for individual compounds may be found in Supporting
1
Information. H NMR spectra were recorded at ambient temperature
A novel, imidazo-pyrrolopyridine hinge-binding scaffold was
identified which appeared to possess an innate preference for
JAK1 over the other JAK isoforms. While the selectivities
quoted are undeniably subtle, they are nonetheless consistent,
with virtually all of the compounds within this series showing
some degree of JAK1 preference, in contrast to the structurally
related, bicyclic inhibitor 1. Since such a profile was un-
expected, attention was taken to ensure accuracy of reported
data. As noted elsewhere, all quoted biochemical and cellular
data are the mean of at least three separate experiments;
furthermore, on average, the coefficient of variation was less
than 0.35 and 0.5 times the mean for biochemical and cell
using Varian Unity Inova or Bruker Avance DRX400 instruments
operating at the indicated frequencies. Chemical shifts were expressed
in ppm relative to an internal standard, tetramethylsilane (ppm = 0.00).
The following abbreviations were used: br = broad signal, s = singlet,
d = doublet, dd = doublet of doublets, t = triplet, q = quartet, p =
pentet, and m = multiplet. Microwave experiments were carried out
using a Biotage Initiator 2.0 (400 W MAGNETRON) which uses a
single-mode resonator and dynamic field tuning.
Strong cation exchange chromatography was performed using Biotage
Isolute SCX-2 columns. Silica gel chromatography was performed using
medium-pressure liquid chromatography (MPLC) on a CombiFlash
Companion (Teledyne Isco) with RediSep normal phase silica gel (35−
60 μm) columns and UV detection at 254 nm.
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a
N-terminus with 5-carboxyfluorescein using the same Caliper LabChip
technology. To determine inhibition constants (Ki), compounds were
diluted serially in DMSO and added to 50 μL kinase reactions
containing 1.5 nM purified JAK1, 0.2 nM purified JAK2, 5 nM purified
JAK3, or 1 nM purified TYK2 enzyme, 100 mM HEPES buffer (pH
7.2), 0.015% Brij-35, 1.5 μM peptide substrate, 25 μM ATP (5 μM
ATP in the case of JAK3), 10 mM MgCl2, and 4 mM DTT at a final
DMSO concentration of 2%. Reactions were incubated at 22 °C in
384-well polypropylene microtiter plates for 30 min and then stopped
by the addition of 25 μL of an EDTA containing solution (100 mM
HEPES buffer pH 7.2, 0.015% Brij-35, 150 mM EDTA), resulting in a
final EDTA concentration of 50 mM. After termination of the kinase
reaction, the proportion of phosphorylated product was determined as
a fraction of total peptide substrate using the Caliper LabChip 3000
according to the manufacturer’s specifications. Ki values were then
determined using the Morrison tight binding model.47
Scheme 4. Preparation of 4-Cyclohexylamine Analogues
JAK Cellular Assays. The activities of compounds were deter-
mined in cell-based assays that are designed to measure JAK-dependent
STAT phosphorylation. TF-1 cells were obtained from the American
Type Culture Collection (ATCC; Manassas, VA). TF-1 cells were
starved overnight in OptiMEM medium with 0.5% Charcoal/Dextran
stripped fetal bovine serum (FBS), 0.1 mM nonessential amino acids
(NEAA), 1 mM sodium pyruvate, and without phenol red at 37 °C.
Compounds were serially diluted in DMSO and incubated for 20 min
at 37 °C with TF-1 cells in 384-well microtiter plates in RPMI-1640 at
a final cell density of 100,000 cells per well and a final DMSO con-
centration of 0.2%. Human recombinant cytokines, IL-6 (30 ng/mL),
or EPO (10 U/mL) were then added at the final concentrations indi-
cated to the microtiter plates containing the TF-1 cells, and com-
pound and the plates were incubated for 30−45 min at 37 °C.
Compound-mediated effects on STAT3 (IL-6) or STAT5 (EPO)
phosphorylation were then measured in the lysates of cells using the Meso
Scale Discovery (MSD) technology (Gaithersburg, Maryland) according
to the manufacturer’s protocol. EC50 values were determined as the
concentration of compound required for 50% inhibition of STAT
phosphorylation relative to that measured for the DMSO control.
4-Chloro-1-phenylsulfonyl-1H-pyrrolo[2,3-b]pyridine (51). A
stirred suspension of 4-chloro-7-azaindole (50; 1.00 g, 6.55 mmol) in
(DCM) 50 mL was treated with DMAP (80.0 mg, 0.66 mmol),
triethylamine (1.36 mL, 9.83 mmol), and benzenesulfonyl chloride
(0.93 mL, 7.21 mmol) at ambient temperature. The mixture was left to
stand overnight. After dilution with DCM, the reaction mixture was
washed with 1 M aqueous HCl solution, saturated sodium hydrogen
carbonate solution, water, and brine, dried with sodium sulfate, and
concentrated under vacuum to give a brown solid. Trituration (diethyl
ether) afforded 1.59 g (83%) of 51 as a beige solid. LCMS (Method 2,
a
Reagents and conditions: (i) 52, EtN(i-Pr)2, i-PrOH, reflux, 1 h, 80%;
(ii) Fe, NH4Cl, MeOH, H2O, reflux, 80%; (iii) (EtO)3CH, p-TsOH
(cat.), PhMe, reflux, 88%; (iv) NaOH, MeOH, THF, rt, 81%; (v) TFA,
DCM, rt, 1 h, 95%; (vi) 62, isobutyryl chloride, EtN(i-Pr)2, DMAP,
DCM, rt, then NaOH, MeOH, rt, 30%; (vii) 62, cyclopropylcarbox-
aldehyde, Na(OAc)3BH, MeOH, rt, then NaOH, MeOH, rt, 44%; (viii)
35, acrylonitrile, EtOH, reflux, 36%; (ix) 35, 3-cyanobenzaldehyde,
Na(OAc)3BH, AcOH, mol. sieves, DCE, rt, 24%; (x) 35, MeSO2Cl,
NEt3, DCM, rt, 38%; (xi) 62, 4-nitrophenyl chloroformate, pyridine,
0 °C, then NaOMe, MeOH, 100 °C in microwave, 10 min, 24%; (xii)
62, Ac2O, HCO2H, THF, then BH3-THF, THF, 80 °C 86%; (xiii)
methyl chloroformate, EtN(i-Pr)2, DCM, then NaOH, THF, MeOH,
H2O, rt, 55%.
1
ESI): RT = 4.48 min, m+H = 293.3. H NMR (400 MHz, CDCl3) δ:
8.31 (d, J = 5.3 Hz, 1 H), 8.18 (m, 2 H), 7.8 (d, J = 4.1 Hz, 1 H), 7.58 (m,
1 H), 7.49 (m, 1 H), 7.20 (d, J = 5.3 Hz, 1 H), 6.71 (d, J = 4.1 Hz, 1 H).
4-Chloro-5-nitro-1-phenylsulfonyl-1H-pyrrolo[2,3-b]pyridine
(52). Tetrabutylammonium nitrate (381 mg, 1.25 mmol) dissolved
in DCM (5 mL) was added dropwise to a stirred solution of 51
(293 mg, 1.00 mmol) in DCM (5 mL) at −5 °C. Trifluoroacetic
anhydride (180 μL, 1.29 mmol) was added while maintaining the
reaction temperature below 0 °C. The mixture was then stirred at
−5 °C for 30 min and ambient temperature for 5 h. TLC indicated
incomplete reaction; therefore, 0.25 equiv each of tetrabutylammo-
nium nitrate and trifluoroacetic anhydride were added and the
resulting mixture left to stand for 18 h at ambient temperature. DCM
was added, and the mixture washed with water, dried with sodium sulfate,
and concentrated under vacuum to give a yellow solid. Purification by
column chromatography on silica gel (gradient: 0 to 25% ethyl acetate in
cyclohexane) gave 266 mg (79%) of 52 as a white solid. LCMS (Method
2, ESI): RT = 4.57 min, m+H = 338.4. 1H NMR (400 MHz, CDCl3) δ:
9.00 (s, 1 H), 8.21 (m, 2 H), 7.94 (d, J = 4.1 Hz, 1 H), 7.66 (t, J = 7.5 Hz,
1 H), 7.55 (t, J = 7.8 Hz, 2 H), 6.87 (d, J = 4.1 Hz, 1 H).
Reverse phase high performance liquid chromatography (HPLC)
was used to purify compounds where indicated. Unless otherwise
indicated, the conditions were elution on a Phenomenex Gemini C18
column (250 × 21.2 mm, 5 μm) as stationary phase and using mobile
phase indicated, operating at a 18 mL/min flow rate using a Gilson
UV/vis −155 dual channel detector and Gilson GX-271 automated
liquid handler.
JAK Enzyme Assays. The activity of the isolated JAK1, JAK2, or
TYK2 kinase domain was measured by monitoring the phosphor-
ylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-
Phe-Arg-Leu-Thr-Thr) fluorescently labeled on the N-terminus with
5-carboxyfluorescein using the Caliper LabChip technology (Caliper
Life Sciences, Hopkinton, MA). Alternatively, the activity of the
isolated JAK3 kinase domain was measured by monitoring the
phosphorylation of a different peptide derived from JAK3 (Leu-Pro-
Leu-Asp-Lys-Asp-Tyr-Tyr-Val-Val-Arg) fluorescently labeled on the
(
)-cis-N-[4-Methyl-1-(phenylmethyl)-piperidin-3-yl]-5-
nitro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-4-amine (54a).
A mixture of 52 (692 mg, 2.05 mmol), ( )-cis-4-methyl-1-(phenylmethyl)-
piperidin-3-ylamine (460 mg, 2.25 mmol), and N,N-diisopropylethylamine
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a
Scheme 5. Preparation of Morpholine Derivatives 37 and 38
a
Reagents and conditions: (i) 52, EtN(i-Pr)2, i-PrOH, reflux, 2 h, quant.; (ii) Fe, NH4Cl, EtOH, H2O, reflux, 2 h, 52%; (iii) (EtO)3CH, AcOH,
110 °C, 0. 25 h, then rt, 82%; (iv) Dess−Martin periodinane, DCM, 0 °C-rt, 89%; (v) morpholine, Na(OAc)3BH, AcOH, THF, rt, 20 h, then
chromatographic separation, trans: 19%, cis: 23%; (vi) NaOH, MeOH, H2O, trans, 67%.
a
Scheme 6. Preparation of Cyclopentylamine Analogues
a
Reagents and conditions: (i) Pd on C, H2, EtOH, 97%; (ii) Raney Ni, H2, EtOH, 75%; (iii) diisopropyl azodicarboxylate, PPh3, AcOH, THF, 0 °C-rt,
then NaOH, MeOH, THF, H2O, rt, 76% 2 steps; (iv) MsCl, Et3N, acetone, 0 °C, 98%; (v) NaN3, DMF, H2O, 100 °C, 77−97%; (vi) PPh3, THF, H2O,
55 °C, 87−91%; (vii) 52, EtN(i-Pr)2, i-PrOH, 120 °C in microwave, 10 min, 87%-quant. ; (viii) Fe, NH4Cl, EtOH, H2O, reflux, 70%; (ix) (EtO)3CH,
p-TsOH (cat.), PhMe, reflux, 77%; (x) TFA, DCM, rt, 86%; (xi) MeO2CCl, EtN(i-Pr)2, DCM, quant.; (xii) NaOH, THF, MeOH, H2O, 89%; (xiii)
SFC resolution.
(1.25 mL, 7.2 mmol) in 2-propanol was heated at 95 °C for 2 h. The
reaction mixture was cooled, and the yellow precipitate that formed
was isolated by filtration and dried in vacuo to afford 940 mg (90%) of
54a. LCMS (Method 3, ESI): RT = 2.61 min, m+H = 506.2.
(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-4,5-diamine (420 mg,
0.90 mmol), triethyl orthoformate (375 μL, 2.25 mmol), and
p-toluenesulfonic acid monohydrate (17.0 mg, 90 μmol) in toluene
(25 mL) was heated at reflux for 16 h. After cooling, ethyl acetate
was added and the mixture washed with saturated aqueous sodium
hydrogen carbonate, water and brine, dried with sodium sulfate, and
concentrated under vacuum. Purification by column chromatography
on silica gel (gradient: 0 to 4% [2 M ammonia in methanol] in DCM)
afforded 356 mg (81%) of ( )-cis-1-[4-methyl-1-(phenylmethyl)-
piperidin-3-yl]-6-(phenylsulfonyl)-1,6-dihydro-imidazo[4,5-d]pyrrolo-
[2,3-b]pyridine as a white foam. LCMS (Method 3, ESI): RT = 2.61 min,
m+H = 486.1.
Step 3: ( )-cis-1-[4-Methyl-1-(phenylmethyl)-piperidin-3-yl]-1,6-
dihydro-imidazo[4,5-d]pyrrolo[2,3-b]pyridine (6). A mixture of
( )-cis-1-[4-methyl-1-(phenylmethyl)-piperidin-3-yl]-6-(phenylsulfon-
yl)-1,6-dihydro-imidazo[4,5-d]pyrrolo[2,3-b]pyridine (356 mg, 0.73
mmol) and 1 M aqueous sodium hydroxide (3.65 mL, 3.65 mmol) in
methanol/THF was stirred at room temperature for 64 h. The reaction
mixture was concentrated under vacuum, and the residue obtained
partitioned between saturated sodium bicarbonate solution and DCM.
The aqueous phase was extracted twice with DCM. The combined
extracts were washed with brine, dried (sodium sulfate), and concen-
trated under vacuum to afford 223 mg (89%) of 6 as a cream colored
( )-cis-1-[4-Methyl-1-(phenylmethyl)-piperidin-3-yl]-1,6-di-
hydro-imidazo[4,5-d]pyrrolo[2,3-b]pyridine (6). Step 1: ( )-cis-
N4-[4-Methyl-1-(phenylmethyl)-piperidin-3-yl]-1-(phenylsulfonyl)-
1H-pyrrolo[2,3-b]pyridine-4,5-diamine. A mixture of 54a (940 mg,
1.86 mmol), iron powder (∼325 mesh, 415 mg, 7.44 mmol), and
ammonium chloride (597 mg, 11.16 mmol) in a methanol/water
mixture (3:1, 64 mL) was heated at reflux for 4 h. The cooled mixture
was filtered through Celite and washed with methanol. The filtrate was
concentrated under vacuum and the residue obtained partitioned
between saturated aqueous sodium bicarbonate and DCM. The
aqueous phase was extracted twice with DCM. The combined extracts
were washed with brine, dried (sodium sulfate), and concentrated
under vacuum. Purification by column chromatography on silica gel
(gradient: 0 to 5% [2 M ammonia in methanol] in DCM) afforded
620 mg (70%) of ( )-cis-N4-[4-methyl-1-(phenylmethyl)-piperidin-3-
yl]-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-4,5-diamine as a
white foam. LCMS (Method 3, ESI): RT = 2.16 min, m+H = 476.1.
Step 2: ( )-cis-1-[4-Methyl-1-(phenylmethyl)-piperidin-3-yl]-6-
(phenylsulfonyl)-1,6-dihydro-imidazo[4,5-d]pyrrolo[2,3-b]pyridine.
A mixture of ( )-cis-N4-[4-methyl-1-(phenylmethyl)-piperidin-3-yl]-1-
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foam. LCMS (Method 1, ESI): RT 2.39 min, m+H = 346.3. 1H NMR
(400 MHz, DMSO-d6) δ: 11.78 (s, 1 H), 8.91 (s, 1 H), 8.56 (s, 1 H),
7.42 (t, J = 3.0 Hz, 1 H), 7.32−7.34 (m, 4 H), 7.22−7.24 (m, 1 H),
6.87 (dd, J = 3.4, 1.9 Hz, 1 H), 5.04 (d, J = 4.0 Hz, 1 H), 3.56−3.57
(m, 2 H), 3.02−3.06 (m, 2 H), 2.63 (dd, J = 12.1, 3.2 Hz, 1 H), 2.16−
2.22 (m, 2 H), 1.53−1.61 (m, 2 H), 0.54 (d, J = 6.8 Hz, 3 H).
hydrate (13 mg, 70 μmol) in toluene (10 mL) was heated to reflux
for 2 h. After cooling, ethyl acetate was added and the mixture
washed with saturated sodium hydrogen carbonate solution, water,
and brine, dried with sodium sulfate, and concentrated under
vacuum. Purification by column chromatography on silica gel
(gradient: 0 to 10% MeOH in DCM) afforded 360 mg (100%) of 1-
[1-(phenylmethyl)-piperidin-4-yl]-6-(phenylsulfonyl)-1,6-dihydro-
imidazo[4,5-d]pyrrolo[2,3-b]pyridine as an orange/yellow residue.
(
)-cis-3-(Imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)-4-
methylpiperidin-1-yl)-3-oxopropanenitrile (4). Step 1: ( )-cis-1-
(4-Methyl-piperidin-3-yl)-1,6-dihydro-imidazo[4,5-d]pyrrolo[2,3-b]-
pyridine. A mixture of 6 (180 mg, 0.52 mmol), palladium hydroxide
(20 wt % on carbon, 37 mg, 0.05 mmol), and ammonium formate
(328 mg, 5.2 mmol) in methanol was heated at 60 °C for 24 h. The
cooled mixture was filtered through Celite and the filter cake washed
with methanol. The filtrate was concentrated under vacuum to afford
110 mg (82%) of ( )-cis-1-(4-methyl-piperidin-3-yl)-1,6-dihydro-
imidazo[4,5-d]pyrrolo[2,3-b]pyridine as an off-white solid. LCMS
(Method 3, ESI): RT 0.35 min, m+H = 255.9.
1
LCMS (Method 2, ESI): RT = 3.05 min, m+H = 472.5; H NMR
(400 MHz, CDCl3) δ: 8.91 (s, 1 H), 8.22 (m, 2 H), 8.01 (br s, 1 H),
7.81 (d, J = 4.0 Hz, 1 H), 7.55 (m, 1 H), 7.47 (m, 2 H), 7.37 (m, 5
H), 6.83 (m, 1 H), 4.43 (m, 1 H), 3.62 (m, 2 H), 3.14 (m, 2 H),
2.21 (m, 6 H).
Step 3: 1-[(1-Phenylmethyl)-piperidin-4-yl]-1,6-dihydro-imidazo-
[4,5-d]pyrrolo[2,3-b]pyridine (12). A solution of 1-[1-(phenylmethyl)-
piperidin-4-yl]-6-(phenylsulfonyl)-1,6-dihydro-imidazo[4,5-d]pyrrolo-
[2,3-b]pyridine (355 mg, 750 μmol) in methanol (18 mL) was treated
with 1 M sodium hydroxide solution (8 mL) and left to stand at
ambient temperature for 18 h. The mixture was partially concentrated
under vacuum, and the resulting suspension was extracted twice with
ethyl acetate. The combined organic extracts were washed with brine,
dried with sodium sulfate, and concentrated under vacuum. Purifi-
cation by column chromatography on silica gel (gradient: 0−10%
methanol in DCM) afforded 189 mg (76%) of 12 as an off-white solid.
Step 2: ( )-cis-3-(Imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)-4-
methylpiperidin-1-yl)-3-oxopropanenitrile (4). A mixture of ( )-cis-
4-methyl-piperidin-3-yl)-1,6-dihydro-imidazo[4,5-d]pyrrolo[2,3-b]-
pyridine (110 mg, 0.43 mmol), cyanoacetic acid (40 mg, 0.47 mmol),
HOBt (87 mg, 0.65 mmol), EDCI (167 mg, 0.86 mmol), and DMAP
(105 mg, 0.86 mmol) in DCM was stirred at room temperature for
18 h. The reaction mixture was evaporated to dryness and purified
by column chromatography on silica gel (gradient: 0 to 6% [2 M
ammonia in methanol] in DCM) to give 43 mg (31%) of 4 as an
off-white solid. Further purification by HPLC (gradient: 5−40%
acetonitrile containing 0.1% v/v aqueous ammonia in 0.1% v/v
aqueous ammonia) afforded 15 mg (11%) of 4 as a white solid. LCMS
1
LCMS (Method 1, ESI): RT = 1.92 min, m+H = 332.2; H NMR
(400 MHz, DMSO-d6) δ: 11.85 (br s, 1 H), 8.58 (s, 1 H), 8.31 (s,
1 H), 7.48 (t, J = 3.0 Hz, 1 H), 7.36 (m, 4 H), 7.27 (m, 1 H), 6.75 (dd,
J = 3.1, 1.9 Hz, 1 H), 4.57 (m, 1 H), 3.59 (s, 2 H), 3.01 (d, J = 11.3 Hz,
2 H), 2.31 (m, 2 H), 2.14 (m, 4 H).
1
(Method 1, ESI): RT 2.14 min, m+H = 323.3. H NMR (400 MHz,
1-Piperidin-4-yl-1,6-dihydro-imidazo[4,5-d]pyrrolo[2,3-b]-
pyridine (13). A stirred mixture of 12 (2.68 g, 8.09 mmol),
ammonium formate (5.09 g, 80.9 mmol), and palladium hydroxide
(20 wt % on carbon, 565 mg, 0.809 mmol) in methanol (150 mL) was
heated at reflux for 5 h. Additional ammonium formate (5.09 g, 80.9
mmol) was added and heating continued for 3 h. After cooling, the
mixture was filtered through Celite and then concentrated in vacuo.
Purification by column chromatography on silica gel (gradient: 0 to
15% [2 M ammonia in methanol] in DCM) and subsequent trituration
(diethyl ether) afforded 1.02 g (52%) of 13 as a beige solid. LCMS
(Method 1, ESI): RT = 0.69 min, m+H = 242.2; 1H NMR (400 MHz,
DMSO-d6) δ: 11.84 (s, 1 H), 8.58 (s, 1 H), 8.27 (s, 1 H), 7.46 (t, J =
2.9 Hz, 1 H), 6.78 (dd, J = 3.5, 1.8 Hz, 1 H), 4.61 (m, 1 H), 3.12 (m,
2 H), 2.77 (m, 2 H), 2.09−1.92 (m, 4 H).
1-(1-Methanesulfonyl-piperidin-4-yl)-1,6-dihydro-imidazo-
[4,5-d]pyrrolo[2,3-b]pyridine (19). To a stirred mixture of 13
(50.0 mg, 0.21 mmol) in DCM (2 mL), methanesulfonyl chloride
(17.0 μL, 0.22 mmol) and triethylamine (57.0 μL, 0.41 mmol) were
added sequentially. The mixture was stirred at room temperature for
30 min and then concentrated in vacuo. The resulting residue was
triturated (water and then diethyl ether) and air-dried to afford
37.0 mg (55%) of 19 as a beige solid. LCMS (Method 1, ESI): RT =
2.02 min, m+H = 320.1. 1H NMR (400 MHz, DMSO-d6) δ: 11.86 (s,
1 H), 8.59 (s, 1 H), 8.34 (s, 1 H), 7.49 (t, J = 2.9 Hz, 1 H), 6.83 (dd,
J = 3.1, 1.8 Hz, 1 H), 4.78 (m, 1 H), 3.77 (m, 2 H), 3.16 (m, 2 H),
2.98 (s, 3 H), 2.28−2.12 (m, 4 H).
4-(Imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)-lpiperidin-
1-yl)-propanenitrile (22). A solution of 13 (50 mg, 207 μmol) and
acrylonitrile (27 μL, 414 μmol) in ethanol (5 mL) was heated at reflux
for 5 h. The mixture was concentrated under vacuum and purified by
column chromatography on silica gel (gradient: 0 to 10% in methanol
in DCM) to afford 53 mg (87%) of 22 as a white solid. LCMS
(Method 1, ESI): RT = 0.75 + 0.87 min, m+H = 295.1. 1H NMR (DMSO-
d6) δ: 11.84 (s, 1 H), 8.58 (s, 1 H), 8.32 (s, 1 H), 7.46 (t, J = 3.0 Hz, 1 H),
6.76 (dd, J = 3.4, 1.9 Hz, 1 H), 4.58 (m, 1 H), 3.09 (d, J = 11.4 Hz, 2 H),
2.72 (m, 4 H), 2.37 (td, J = 11.3, 3.3 Hz, 2 H), 2.14 (m, 4 H).
((S)-1-Isopropylpyrrolidin-2-yl-[4-(imidazo[4,5-d]pyrrolo-
[2,3-b]pyridin-1(6H)-yl)-piperidin-1-yl]-methanone (26). To a
stirred mixture of (S)-1-isopropylpyrrolidine-2-carboxylic acid (31.2 mg,
0.198 mmol), HATU (76 mg, 0.198 mmol), and N,N-diisopropylethyl-
amine (40 μL, 0.247 mmol) in DMF (2 mL) was added 13 (40 mg,
DMSO-d6) δ: 11.85 (s, 1 H), 8.59−8.60 (m, 1 H), 8.23−8.59 (m,
1 H), 7.46 (t, J = 3.0 Hz, 1 H), 6.87−6.89 (m, 1 H), 5.08 (d, J =
4.7 Hz, 1 H), 4.38−4.40 (m, 2 H), 4.10 (d, J = 18.7 Hz, 1 H), 3.83 (d, J =
20.0 Hz, 1 H), 3.72 (d, J = 14.7 Hz, 1 H), 3.60 (dd, J = 13.8, 3.61 Hz,
1 H), 2.42 (br s, 1 H), 1.71−1.77 (m, 2 H), 0.64 (dd, J = 23.1,
6.83 Hz, 3 H).
N-[1-(Phenylmethyl)-piperidin-4-yl]-5-nitro-1-(phenylsulfon-
yl)-1H-pyrrolo[2,3-b]pyridin-4-amine (54f). A mixture of 52
(300 mg, 890 μmol), 4-amino-1-benzyl piperidine (203 mg, 1.07 mmol),
and diisopropylethylamine (229 μL, 1.34 mmol) in 2-propanol (5 mL)
was heated in a microwave reactor at 120 °C for 12 min. The mixture
was diluted with DCM and then purified by column chromatography
on silica gel (gradient: 0 to 100% ethyl acetate in cyclohexane) to
afford 449 mg (100%) of 54f as a yellow residue. LCMS (Method 2,
1
ESI): RT = 3.35 min, m+H = 492.6. H NMR (400 MHz, CDCl3) δ:
9.10 (s, 1 H), 8.18 (m, 2 H), 7.60 (m, 2 H), 7.51 (m, 2 H), 7.33 (m,
5 H), 6.69 (d, J = 4.2 Hz, 1 H), 4.00 (m, 1 H), 3.58 (m, 2 H), 2.85 (m,
2 H), 2.31 (m, 2 H), 2.11 (m, 2 H), 1.79 (m, 2 H).
1-[(1-Phenylmethyl)-piperidin-4-yl]-1,6-dihydro-imidazo-
[4,5-d]pyrrolo[2,3-b]pyridine (12). Step 1: N4-[1-(Phenylmethyl)-
piperidin-3-yl]-1-phenylsulfonyl-1H-pyrrolo[2,3-b]pyridine-4,5-dia-
mine. A mixture of 54f (442 mg, 890 μmol), iron powder (201 mg,
3.60 mmol), and ammonium chloride (289 mg, 5.39 mmol) in methanol/
water (12 mL, 3: 1) was heated at reflux for 4 h. After cooling, the
mixture was filtered through Celite, thoroughly washing the filter cake
with methanol. The filtrate and washings were combined and con-
centrated under vacuum. The resulting residue was partitioned be-
tween DCM and water, the organic layer dried with sodium sulfate and
concentrated under vacuum. Purification by column chromatography
on silica gel (gradient: 0 to 10% methanol in DCM) afforded 321 mg
(77%) of N4-[1-(phenylmethyl)-piperidin-3-yl]-1-phenylsulfonyl-1H-
pyrrolo[2,3-b]pyridine-4,5-diamine as an off-white foam. LCMS
1
(Method 2, ESI): RT = 2.55−2.68 min, m+H = 462.5. H NMR
(400 MHz, CDCl3) δ: 8.12 (m, 2 H), 7.83 (s, 1 H), 7.52 (m, 1 H),
7.44 (m, 3 H), 7.34 (m, 5 H), 6.51 (d, J = 4.2 Hz, 1 H), 4.75 (m, 1 H),
3.69 (m, 3 H), 2.88 (m, 4 H), 2.29 (m, 2 H), 2.06 (m, 2 H), 1.71 (m, 2 H).
Step 2: 1-[1-(Phenylmethyl)-piperidin-4-yl]-6-(phenylsulfonyl)-
1,6-dihydro-imidazo[4,5-d]pyrrolo[2,3-b]pyridine. A mixture of
N4-[1-(phenylmethyl)-piperidin-3-yl]-1-phenylsulfonyl-1H-pyrrolo-
[2,3-b]pyridine-4,5-diamine (310 mg, 670 μmol), triethyl ortho-
formate (279 μL, 1.68 mmol), and p-toluenesulfonic acid mono-
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0.165 mmol), and the resulting mixture was stirred at ambient tem-
perature for 1 h. The reaction mixture was concentrated and purified by
column chromatography on silica gel (gradient: 0 to 10% [2 M
ammonia in methanol] in DCM) to afford 55 mg (88%) of 26. LCMS
(Method 1, ESI): RT = 1.57 min, m+H = 381.16. 1H NMR (400 MHz,
DMSO-d6) δ: 11.85 (s, 1 H), 8.57 (s, 1 H), 8.30 (s, 1 H), 7.40 (t, J = 2.9
Hz, 1 H), 6.73 (d, J = 3.3 Hz, 1 H), 4.84 (t, J = 4.1 Hz, 1 H), 4.61 (d,
J = 13.6 Hz, 3 H), 2.50−2.45 (m, 4 H), 2.20 (d, J = 12.6 Hz, 3 H), 2.07
(d, J = 3.8 Hz, 1 H), 2.01 (d, J = 12.5 Hz, 3 H), 1.81 (m, 4 H), 1.79 (t,
J = 3.6 Hz, 2 H), 1.02 (dd, J = 22.8, 6.42 Hz, 1 H).
4-Benzyloxycarbonylamino-4-methyl-piperidine-1-carbox-
ylic Acid tert-Butyl Ester (56). Compound 55 (1.0 g, 4.11 mmol),
triethylamine (830 μL, 5.96 mmol), and diphenylphosphoroyl azide
(1.2 mL, 5.59 mmol) in toluene were stirred at room temperature for
1 h. Benzyl alcohol (2.15 mL, 20.55 mmol) was added, and the
reaction stirred at 80 °C for 18 h. The reaction mixture was cooled and
concentrated under vacuum. Purification by column chromatography
on silica gel (gradient: 0 to 25% ethyl acetate in cyclohexane) afforded
1.79 g of 56 (contaminated with benzyl alcohol). The material was
used in the next step without further purification. LCMS (Method 3,
ESI): RT = 3.86 min, m+H = 349.0.
and concentrated under vacuum. Purification by column chromatog-
raphy on silica gel (gradient: 0 to 4% [2 M ammonia in methanol] in
DCM) afforded 630 mg (86%) of 4-[(6-phenylsulfonyl-4-(imidazo[4,5-d]-
pyrrolo[2,3-b]pyridin-1(6H)-yl)]-4-methyl-piperidine-1-carboxylic
acid tert-butyl ester as an off-white foam. LCMS (Method 2, ESI): RT =
3.54 min, m+H = 496.2.
Step 4: 4-Methyl-4-(imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-
yl)-piperidine-1-carboxylic Acid tert-Butyl Ester (58). A mixture of
4-[(6-phenylsulfonyl-4-(imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-
yl)]-4-methyl-piperidine-1-carboxylic acid tert-butyl ester (630 mg,
1.27 mmol) and 1 M aqueous sodium hydroxide (6.36 mL, 6.36 mmol)
in methanol/THF was stirred at room temperature for 60 h. The
reaction mixture was concentrated under vacuum and the residue
obtained partitioned between saturated sodium bicarbonate solution
and DCM. The aqueous phase was extracted twice with DCM. The
combined extracts were washed with brine, dried (sodium sulfate), and
concentrated under vacuum to afford 440 mg (98%) of 58 as an off-
white foam. LCMS (Method 1, ESI): RT 3.10 min, m+H = 356.2. 1H
NMR (400 MHz, DMSO-d6) δ: 11.92 (s, 1 H), 8.62 (s, 1 H), 8.34 (s,
1 H), 7.45 (t, J = 3.1 Hz, 1 H), 6.62 (dd, J = 3.5, 1.9 Hz, 1 H), 3.71 (m,
2 H), 3.35 (br s, 2 H), 2.52−2.55 (m, 1 H), 2.04 (d, J = 13.5 Hz, 3 H),
1.85 (s, 3 H), 1.45 (s, 9 H).
1-(4-Methyl-piperidin-4-yl)-1,6-dihydro-imidazo[4,5-d]-
pyrrolo[2,3-b]pyridine (28). A solution of 58 (420 mg, 1.18 mmol)
and TFA (7 mL) in DCM (28 mL) was stirred at room temperature
for 1 h. The reaction mixture was concentrated under vacuum and a
portion purified by column chromatography on silica gel (gradient:
0 to 10% [2 M ammonia in methanol] in DCM) to afford 77 mg of 28
as a white solid. LCMS (Method 1, ESI): RT 1.21 min, m+H = 278.2.
1H NMR (400 MHz, DMSO-d6) δ: 11.86 (s, 1 H), 8.61 (s, 1 H), 8.31
4-Amino-4-methyl-piperidine-1-carboxylic Acid tert-Butyl
Ester (57). A solution of 56 (1.8 g, 5.2 mmol) in ethanol was
purged with argon, palladium on carbon (10% Pd content, 550 mg,
0.52 mmol) added, and the reaction mixture stirred under an atmo-
sphere of hydrogen for 18 h. The solids were removed by filtration
through Celite washing with ethanol and the filtrate concentrated
under vacuum. Purification by column chromatography on silica gel
(gradient: 0 to 5% [2 M ammonia in methanol] in DCM) afforded
525 mg (47%) of 57 as a clear oil. LCMS (Method 3, ESI): RT = 0.31 min,
1
m+H = 214.9; H NMR (400 MHz, CDCl3) δ: 3.40−3.42 (m, 4 H),
(s, 1 H), 7.47 (dd, J = 3.4, 2.6 Hz, 1 H), 7.01 (dd, J = 3.5, 1.9 Hz, 1 H),
2.82−2.85 (m, 4 H), 2.55 (dd, J = 13.5, 6.9 Hz, 2 H), 2.37 (br s, 1 H),
1.93−1.97 (m, 2 H), 1.81 (s, 3 H).
1.48−1.51 (m, 2 H), 1.46 (s, 9 H), 1.36−1.39 (m, 2 H), 1.14 (s, 3 H).
4-Methyl-4-(imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)-
piperidine-1-carboxylic Acid tert-Butyl Ester (58). Step 1: 4-(5-
Nitro-1-phenylsulfonyl-1H-pyrrolo[2,3-b]pyridine-4-ylamino)-4-methyl-
piperdine-1-carboxylic Acid tert-Butyl Ester. A mixture of 52 (750 mg,
2.23 mmol), 57 (525 mg, 2.45 mmol), and N,N-diisopropylethylamine
(1.36 mL, 7.80 mmol) in 2-propanol was heated at 95 °C for 64 h. The
reaction mixture was cooled and the precipitate recovered by filtration.
The solid was purified by column chromatography on silica gel
(gradient: 0 to 30% ethyl acetate in cyclohexane) to afford 0.91 g
(79%) of 4-(5-nitro-1-phenylsulfonyl-1H-pyrrolo[2,3-b]pyridine-4-yla-
mino)-4-methyl-piperdine-1-carboxylic acid tert-butyl ester as a yellow
foam. LCMS (Method 3, ESI): RT = 4.35 min, m+H = 516.2.
Step 2: 4-(5-Amino-1-phenylsulfonyl-1H-pyrrolo[2,3-b]pyridine-
4-ylamino)-4-methyl-piperdine-1-carboxylic Acid tert-Butyl Ester.
A mixture of 4-(5-nitro-1-phenylsulfonyl-1H-pyrrolo[2,3-b]pyridine-
4-ylamino)-4-methyl-piperdine-1-carboxylic acid tert-butyl ester
(910 mg, 1.76 mmol), iron powder (∼325 mesh, 393 mg, 7.04 mmol)
and ammonium chloride (565 mg, 10.56 mmol) in a methanol/water
mixture (3:1, 48 mL) was heated at 85 °C for 4 h. The cooled mixture
was filtered through Celite washing with methanol. The filtrate was
concentrated under vacuum and the residue obtained partitioned
between saturated sodium bicarbonate solution and DCM. The aqueous
phase was extracted twice with DCM. The combined extracts were
washed with brine, dried (sodium sulfate), and concentrated under
vacuum to afford a pink residue. Purification by column chromatography
on silica gel (gradient: 0 to 4% [2 M ammonia in methanol] in DCM)
afforded 720 mg (84%) of 4-(5-amino-1-phenylsulfonyl-1H-pyrrolo[2,3-b]-
pyridine-4-ylamino)-4-methyl-piperdine-1-carboxylic acid tert-butyl
ester as an off-white foam. LCMS (Method 2, ESI): RT = 3.21 min,
m+H = 486.3.
1-(1-Methanesulfonyl-4-methyl-piperidin-4-yl)-1,6-dihydro-
imidazo[4,5-d]pyrrolo[2,3-b]pyridine (30). A mixture of 28
(136 mg, 0.53 mmol), methanesulfonyl chloride (41.5 μL, 0.53 mmol),
and triethylamine (88.5 μL, 0.64 mmol) in DCM was stirred at room
temperature for 1.5 h. The reaction mixture was washed with
saturated sodium hydrogen carbonate solution and brine, dried with
sodium sulfate, and concentrated under vacuum. Purification by
column chromatography on silica gel (gradient: 0 to 6% [2 M
ammonia in methanol] in DCM) afforded 102 mg (58%) of 30 as a
white solid. LCMS (Method 1, ESI): RT 2.19 min, m+H = 334.3.
1H NMR (400 MHz, DMSO-d6) δ: 11.93 (s, 1 H), 8.63 (s, 1 H),
8.34 (s, 1 H), 7.52 (t, J = 3.1 Hz, 1 H), 6.79 (dd, J = 3.5, 1.9 Hz, 1 H),
3.32 (m, 6 H, includes water solvent peak), 2.92 (s, 3 H), 2.73 (m, 2 H),
2.19 (m, 2 H), 1.85 (s, 3 H).
trans-[4-(5-Nitro-1-phenylsulfonyl-1H-pyrrolo[2,3-b]-
pyridine-4-ylamino)-cyclohexyl]-carbamic Acid tert-Butyl Ester
(60). A mixture of 52 (1.01 g, 3.0 mmol), 59 (707 mg, 3.30 mmol),
and N,N-diisopropylethylamine (1.80 mL, 10.5 mmol) in 2-propanol
was heated at 95 °C for 1 h. The cooled reaction mixture was
concentrated under vacuum to afford a yellow residue. Purification by
column chromatography on silica gel (gradient: 0 to 10% ethyl acetate
in DCM) afforded 1.24 g (80%) of 60 as a yellow foam. LCMS
(Method 3, ESI): RT = 4.13 min, m+H = 516.3.
trans-[4-(6-Phenylsulfonyl-4-(imidazo[4,5-d]pyrrolo[2,3-b]-
pyridin-1(6H)-yl)-cyclohexyl]-carbamic Acid tert-Butyl Ester
(61). Step 1: trans-[4-(5-Amino-1-phenylsulfonyl-1H-pyrrolo[2,3-
b]pyridine-4-ylamino)-cyclohexyl]-carbamic Acid tert-Butyl Ester.
A mixture of 60 (1.14 g, 2.21 mmol), iron powder (∼325 mesh,
494 mg, 8.84 mmol), and ammonium chloride (709 mg, 13.26 mmol)
in a methanol/water mixture (3:1, 44 mL) was heated at reflux for 4 h.
The cooled mixture was filtered through Celite washing with methanol.
The filtrate was concentrated under vacuum and the residue obtained
partitioned between saturated sodium bicarbonate solution and DCM.
The aqueous phase was extracted twice with DCM. The combined
extracts were washed with brine, dried (sodium sulfate), and concen-
trated under vacuum. Purification by column chromatography on silica
gel (gradient: 0 to 5% methanol in DCM) afforded 940 mg (88%) of
Step 3: 4-[6-Phenylsulfonyl-4-(imidazo[4,5-d]pyrrolo[2,3-b]-
pyridin-1(6H)-yl)]-4-methyl-piperidine-1-carboxylic Acid tert-Butyl
Ester. A mixture of 4-(5-amino-1-phenylsulfonyl-1H-pyrrolo[2,3-b]-
pyridine-4-ylamino)-4-methyl-piperdine-1-carboxylic acid tert-butyl
ester (720 mg, 1.48 mmol), triethyl orthoformate (615 μL, 3.70 mmol),
and p-toluenesulfonic acid monohydrate (28.0 mg, 148 μmol) in
toluene (50 mL) was heated at reflux for 24 h. After cooling, ethyl
acetate was added and the mixture washed with saturated sodium
hydrogen carbonate solution, water and brine, dried with sodium sulfate,
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trans-[4-(5-amino-1-phenylsulfonyl-1H-pyrrolo[2,3-b]pyridine-4-yla-
mino)-cyclohexyl]-carbamic acid tert-butyl ester as an off-white solid.
LCMS (Method 3, ESI): RT = 2.65 min, m+H = 486.4.
(3.50 mL, 17.9 mmol) and the mixture stirred at 0 °C for 10 min.
A solution of acetic acid (2.4 mL, 41.8 mmol) in dry THF (18 mL)
was added dropwise and the mixture stirred at 0 °C for 20 min, then at
ambient temperature overnight. The mixture was concentrated under
vacuum and the residue obtained purified by column chromatography
on silica gel (gradient: 0 to 30% EtOAc in cyclohexane) to afford
2.87 g (79%) of ( )-trans-acetic acid 3-tert-butoxycarbonylamino-
cyclopentyl ester as a white solid. LCMS (Method 2, ESI): RT =
3.23 min, no m+H observed. 1H NMR (400 MHz, CDCl3) δ: 5.18 (m,
1 H), 4.46 (br s, 1 H), 4.14 (m, 1 H), 2.12 (m, 3 H), 2.01 (s, 3 H),
1.69 (m, 2 H), 1.44 (s, 9 H), 1.41 (m, 1 H). Step 2: A solution of the
preceding material (2.27 g, 11.8 mmol) in methanol (6.8 mL) and
THF (6.8 mL) was treated with 4 M aqueous sodium hydroxide solu-
tion (6.8 mL, 27.2 mmol) and the mixture stirred at ambient
temperature for 1.5 h. The mixture was concentrated under vacuum
and the residue obtained partitioned between DCM and brine. The
aqueous phase was extracted twice with DCM, and the combined
extracts were washed with brine, dried (sodium sulfate), and
concentrated under vacuum to afford 2.28 g (96%) of 69 as a white
solid. LCMS (Method 2, ESI): RT = 2.46 min, m+H = 202 (weak),
m+H+MeCN = 243. 1H NMR (400 MHz, CDCl3) δ: 4.45 (br s, 1 H),
4.40 (m, 1 H), 4.17 (m, 1 H), 2.21 (m, 1 H), 2.02 (m, 2 H), 1.62 (m,
2 H), 1.44 (s, 9 H), 1.40 (m, 1 H).
Step 2: trans-[4-(6-phenylsulfonyl-4-(imidazo[4,5-d]pyrrolo[2,3-
b]pyridin-1(6H)-yl)-cyclohexyl]-carbamic Acid tert-Butyl Ester (61).
A mixture of trans-[4-(5-amino-1-phenylsulfonyl-1H-pyrrolo[2,3-b]-
pyridine-4-ylamino)-cyclohexyl]-carbamic acid tert-butyl ester (940 mg,
1.94 mmol), triethyl orthoformate (807 μL, 4.85 mmol), and
p-toluenesulfonic acid monohydrate (37.0 mg, 194 μmol) in toluene
(25 mL) was heated at reflux for 3.5 h. After cooling, ethyl acetate was
added and the mixture washed with saturated sodium hydrogen
carbonate solution, water, and brine, dried with sodium sulfate, and
concentrated under vacuum. Purification by column chromatography
on silica gel (gradient: 0 to 5% [2 M ammonia in methanol] in DCM)
afforded 0.81 g (84%) of 61. LCMS (Method 3, ESI): RT = 3.47 min,
m+H = 496.2. 1H NMR (400 MHz, CDCl3) δ: 8.95 (s, 1 H), 8.23 (dd,
J = 7.8, 1.3 Hz, 3 H), 7.85 (d, J = 4.0 Hz, 1 H), 7.55 (m, 1 H), 7.47−
7.48 (m, 2 H), 6.75 (d, J = 4.0 Hz, 1 H), 4.40−4.47 (m, 2 H), 3.63 (br s,
1 H), 2.33 (m, 5 H), 1.99 (m, 2 H), 1.47 (s, 9 H).
trans-[4-(Imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)-cyclo-
hexyl]-carbamic Acid tert-Butyl Ester (33). A mixture of 61 (810 mg,
1.63 mmol) and 1 M aqueous sodium hydroxide (8.2 mL, 8.2 mmol)
in methanol/THF was stirred at room temperature for 18 h. The
reaction mixture was concentrated under vacuum and the residue
partitioned between saturated sodium bicarbonate solution and DCM. The
aqueous phase was extracted twice with DCM. The combined extracts
were washed with brine, dried (sodium sulfate), and concentrated under
vacuum. Purification by column chromatography on silica gel (gradient: 0
to 6% [2 M ammonia in methanol] in DCM) afforded 470 mg (81%) of
33 as a white solid. LCMS (Method 1, ESI): RT 3.06 min, m+H = 356.2.
1H NMR (400 MHz, DMSO-d6) δ: 11.84 (s, 1 H), 8.57 (s, 1 H), 8.28 (s,
( )-trans-(3-Amino-cyclopentyl)-carbamic Acid tert-Butyl
Ester (70). Step 1: A solution of 68 (2.10 g, 10.4 mmol) in acetone
(44 mL) containing triethylamine (2.91 mL, 20.9 mmol) was cooled
to 0 °C and treated dropwise with methanesulfonyl chloride (1.21 mL,
15.7 mmol). After 10 min, the mixture was concentrated under
vacuum and the residue obtained partitioned between EtOAc and
water. The aqueous phase was extracted twice with ethyl acetate, and
the combined extracts were washed with 10% aqueous citric acid solu-
tion, saturated sodium hydrogen carbonate solution, and brine, dried
(sodium sulfate), and concentrated under vacuum to afford 2.88 g
(99%) of ( )-cis-methanesulfonic acid 3-tert-butoxycarbonylamino-
cyclopentyl ester as a cream colored solid. LCMS (Method 2, ESI):
1 H), 7.47 (t, J = 3.0 Hz, 1 H), 6.89 (d, J = 7.6 Hz, 1 H), 6.68 (dd, J = 3.4,
1.9 Hz, 1 H), 4.51−4.54 (m, 1 H), 3.43 (br s, 1 H), 2.16 (d, J = 11.7 Hz, 2
H), 2.00 (d, J = 12.4 Hz, 4 H), 1.51−1.56 (m, 2 H), 1.41 (s, 9 H).
trans-4-(Imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)-cyclo-
hexylamine (35). A mixture of 33 (442 mg, 1.24 mmol) and TFA
(4 mL) in DCM (16 mL) was stirred at room temperature for 1.5 h.
The reaction mixture was concentrated under vacuum and the residue
purified by chromatography on an SCX-2 column (gradient: 10 to 50%
[2 M ammonia in methanol] in DCM) to afford 300 mg (95%) of 35
as a white solid. LCMS (Method 1, ESI): RT 0.75 min, m+H = 256.1.
1H NMR (400 MHz, DMSO-d6) δ: 11.84 (br s, 1 H), 8.57 (s, 1 H),
1
RT = 2.97 min, m+H = 280 (weak), m+H+MeCN = 343. H NMR
(400 MHz, CDCl3) δ: 5.14 (m, 1 H), 4.72 (br s, 1 H), 4.12 (br s, 1 H),
3.01 (s, 3 H), 2.36 (m, 1 H), 2.11 (m, 2 H), 1.94 (m, 1 H), 1.87 (m,
1 H), 1.70 (m, 1 H), 1.44 (s, 9 H). Step 2: A solution of ( )-cis-
methanesulfonic acid 3-tert-butoxycarbonylamino-cyclopentyl ester
(2.88 g, 10.3 mmol) in DMF (21 mL) was treated with a solution
of sodium azide (0.94 g, 14.5 mmol) in water (3.7 mL), and the
reaction mixture was heated at 100 °C for 1 h. The cooled mixture was
concentrated under vacuum and the residue obtained partitioned
between ethyl acetate and water. The aqueous phase was extracted
twice with ethyl acetate, and the combined extracts were washed with
water and brine, dried (sodium sulfate), and concentrated under
vacuum to afford 2.24 g (96%) of ( )-trans-(3-azido-cyclopentyl)-
carbamic acid tert-butyl ester as a yellow oil. LCMS (Method 2, ESI):
8.26 (s, 1 H), 7.47 (d, J = 3.4 Hz, 1 H), 6.71 (d, J = 3.4 Hz, 1 H),
4.51−4.54 (m, 1 H), 2.73−2.75 (m, 1 H), 2.13 (d, J = 11.7 Hz, 2 H),
1.94−1.98 (m, 4 H), 1.40−1.43 (m, 2 H).
trans-3-[4-(Imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)-cy-
clohexylamino]-propionitrile (41). A mixture of acrylonitrile
(64.5 μL, 0.98 mmol) and 35 (50.0 mg, 196 μmol) in ethanol (5.5 mL)
was heated at 80 °C for 1 h. After cooling, the solvent was removed
under vacuum. The resulting residue was triturated (water and then
diethyl ether) and air-dried to afford 22.0 mg (36%) of 41 as a cream
colored solid. LCMS (Method 1, ESI): RT = 1.22 min, m+H = 309.2.
1H NMR (400 MHz, DMSO-d6) δ: 11.83 (s, 1 H), 8.57 (s, 1 H), 8.26
1
RT = 3.48 min, no m+H observed, m+H+MeCN = 290. H NMR
(400 MHz, CDCl3) δ: 4.45 (br s, 1 H), 4.09 (br s, 1 H), 4.03 (m,
1 H), 2.10 (m, 3 H), 1.71 (m, 2 H), 1.44 (m, 10 H). Step 3: A solution
of ( )-trans-(3-azido-cyclopentyl)-carbamic acid tert-butyl ester (2.24 g,
9.9 mmol) in THF (90 mL) was treated with water (9 mL) then
triphenylphosphine (2.86 g, 10.9 mmol), and the reaction mixture was
heated to 55 °C overnight. The cooled mixture was concentrated
under vacuum to a solid residue. The solid was triturated with ether
and filtered, rinsing with ether. The filtrate was concentrated under
vacuum to a yellow oil then triturated with ether as before. The filtrate
was concentrated to an orange oil that was purified by column
chromatography on silica gel (gradient: 0 to 15% methanol in DCM
plus 1% triethylamine) to afford 1.80 g (91%) of 70 as a white paste
that crystallized on standing. LCMS (Method 2, ESI): RT = 0.84 min,
(s, 1 H), 7.46 (t, J = 2.6 Hz, 1 H), 6.73 (dd, J = 3.1, 1.8 Hz, 1 H), 4.57
(m, 1 H), 2.83 (t, J = 6.6 Hz, 2 H), 2.59 (m, 3 H), 2.17 (m, 2 H), 2.06
(m, 2 H), 1.95 (m, 3 H), 1.40 (m, 2 H).
(
)-cis-(3-Hydroxy-cyclopentyl)-carbamic Acid tert-Butyl
Ester (68). A solution of 6743 in ethanol (90 mL) was treated with
10% palladium on carbon (1.0 g, 0.94 mmol palladium) and hydro-
genated for 3 days at ambient pressure. More catalyst was added
(1.2 g, 1.1 mmol palladium), and the mixture was hydrogenated for a
further 24 h. The suspension was filtered through Celite washing with
ethanol. The filtrate was concentrated under vacuum to afford 8.86 g
(97%) of 68 as a dark green oil. LCMS (Method 2, ESI): RT = 3.08 min,
m+H = 200. 1H NMR (400 MHz, CDCl3) δ: 4.71 (m, 1 H), 4.54 (m,
1 H), 1.87 (m, 3 H), 1.68 (m, 3 H), 1.50 (s, 9 H).
1
m+H = 201. H NMR (400 MHz, CDCl3) δ: 4.47 (br s, 1 H), 4.10
(br s, 1 H), 3.46 (m, 1 H), 2.18 (m, 1 H), 2.00 (m, 1 H), 1.72 (t, J =
6.7 Hz, 2 H), 1.44 (s, 9 H), 1.34 (m, 4 H).
( )-trans-(3-Hydroxy-cyclopentyl)-carbamic Acid tert-Butyl
Ester (69). Step 1: A solution of 68 (3.00 g, 14.9 mmol) in dry THF
(81 mL) at 0 °C under an inert atmosphere was treated with triphenyl-
phosphine (4.30 g, 16.4 mmol) and diisopropylazodicarboxylate
( )-trans-[3-(6-Phenylsulfonyl-imidazo[4,5-d]pyrrolo[2,3-b]-
pyridin-1(6H)-yl)-cyclopentyl]-carbamic Acid tert-Butyl Ester
(72). Step 1: A solution of 70 (1.70 g, 8.5 mmol) in 2-propanol (21 mL)
was treated with N,N-diisopropylethylamine (1.60 mL, 9.2 mmol)
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followed by 52 (2.39 g, 7.1 mmol), and the mixture was heated at
120 °C for 10 min using microwave irradiation. The cooled reaction
mixture was filtered, and the solid was rinsed with 2-propanol and
dried under vacuum to afford 3.00 g (84%) of ( )-trans-[3-(5-nitro-1-
phenylsulfonyl-1H-pyrrolo[2,3-b]pyridin-4-ylamino)-cyclopentyl]-car-
bamic acid tert-butyl ester as a yellow crystalline solid. LCMS (Method
2, ESI): RT = 4.09 min, m+H = 502. 1H NMR (400 MHz, CDCl3) δ:
9.09 (s, 1 H), 9.06 (d, J = 7.0 Hz, 1 H), 8.19 (d, J = 8.0 Hz, 2 H), 7.62
(m, 2 H), 7.52 (t, J = 8.0 Hz, 2 H), 6.84 (d, J = 4.2 Hz, 1 H), 4.58
(m, 2 H), 4.11 (m, 1 H), 2.36 (m, 1 H), 2.25 (m, 1 H), 2.10 (m, 2 H),
1.73 (m, 1 H), 1.62 (m, 1 H), 1.45 (s, 9 H). Step 2: A suspension of
( )-trans-[3-(5-nitro-1-phenylsulfonyl-1H-pyrrolo[2,3-b]pyridin-4-yla-
mino)-cyclopentyl]-carbamic acid tert-butyl ester (3.0 g, 6.0 mmol) in
ethanol (22 mL) was treated with water (8 mL), ammonium chloride
(1.92 g, 35.9 mmol), and iron powder (1.34 g, 24.0 mmol) and the
mixture heated at 80 °C for 1.5 h. The cooled reaction mixture was
filtered through Celite washing with ethanol/water. The filtrate was
concentrated under vacuum and the residue obtained partitioned
between DCM and a saturated sodium hydrogen carbonate solution.
The aqueous phase was extracted twice with DCM, and the combined
extracts were washed with brine, dried (sodium sulfate), and con-
centrated under vacuum to afford a beige foam. The foam was
crystallized from DCM to afford 1.45 g (51%) of ( )-cis-[3-(5-amino-
1-phenylsulfonyl-1H-pyrrolo[2,3-b]pyridin-4-ylamino)-cyclopentyl]-
carbamic acid tert-butyl ester as a white solid. LCMS (Method 2, ESI):
RT = 2.85 min, m+H = 472. 1H NMR (400 MHz, CDCl3) δ: 8.13 (d,
J = 8.1 Hz, 2 H), 7.85 (s, 1 H), 7.54 (tt, J = 7.5, 1.3 Hz, 1 H), 7.45 (m,
3 H), 6.62 (d, J = 4.4 Hz, 1 H), 4.99 (br s, 1 H), 4.56 (br s, 1 H), 4.41
(br s, 1 H), 4.16 (br s, 1 H), 2.24 (m, 2 H, 2.07 (br s, 1 H), 1.88 (m,
1 H), 1.63 (m, 1 H), 1.50 (m, 1 H), 1.44 (s, 9 H). The mother liquor
was concentrated under vacuum to a brown foam that was crystallized
from DCM to provide a second crop (0.34 g, 12%). The mother liquors
from the second crop were concentrated under vacuum to a brown foam.
Purification by column chromatography on silica gel (gradient: 0 to 7%
methanol in DCM) afforded a further crop of 0.46 g (16%) of product
as a white solid. Step 3: A suspension of ( )-cis-[3-(5-amino-1-phenyl-
sulfonyl-1H-pyrrolo[2,3-b]pyridin-4-ylamino)-cyclopentyl]-carbamic
acid tert-butyl ester (1.25 g, 2.7 mmol) in toluene (12.5 mL) was
treated with triethyl orthoformate (1.3 mL, 8.0 mmol) followed by
p-toluene sulfonic acid monohydrate (50 mg, 0.27 mmol), and the
reaction mixture was heated to 100 °C for 1.75 h. The cooled reaction
mixture was filtered, and the filter cake was washed with toluene and
water and dried to afford a white solid. Purification by column
chromatography on silica gel (gradient: 0 to 7% methanol in DCM)
afforded 0.90 g (70%) of 72 as a white solid. LCMS (Method 2, ESI):
RT = 3.59 min, m+H = 482. 1H NMR (400 MHz, DMSO-d6) δ: 8.72
(s, 1 H), 8.49 (s, 1 H), 8.13 (d, J = 8.5 Hz, 2 H), 8.01 (d, J = 4.2 Hz,
1 H), 7.70 (tt, J = 7.7, 1.2 Hz, 1 H), 7.61 (t, J = 8.1 Hz, 2 H), 7.29 (d,
J = 4.0 Hz, 1 H), 7.18 (d, J = 4.7 Hz, 1 H), 5.23 (p, J = 7.7 Hz, 1 H),
4.11 (m, 1 H), 2.36 (m, 1 H), 2.29 - 2.08 (m, 3 H), 1.96 (m, 1 H),
1.64 (m, 1 H), 1.40 (s, 9 H).
trans-3-(Imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)-cyclo-
pentyl]-carbamic Acid tert-Butyl Ester (49). Compound 47 was
resolved using SFC (21.2 × 250 mm, 5 μm Chiralpak AD; mobile
phase, 35% methanol/65% CO2; flow rate, 60 mL/min; detection at
230 nm). LCMS (Method 4, ESI): RT = 0.94 min, m = 341.2.
ASSOCIATED CONTENT
* Supporting Information
■
S
Details of crystallographic methods and procedures: analytical
LCMS methods; experimental LogD measurement procedures;
in vitro and in vivo ADME experimental procedures; details of
molecular modeling calculations and methods; preparation and
characterization of 7−11, 14−18, 20, 21, 23−25, 27, 29, 31,
32, 34, 36−40, 42−46, 48, 54b−d, 54g, 62, 64−66, 71, 73,
and 74. This material is available free of charge via the Internet
at http://pubs.acs.org.
Accession Codes
PDB nos.: 4E6D for 7 complexed with JAK2TM, 4E6Q for 12
complexed with JAK2TM, 4E5W for 26 complexed with JAK1,
4E4L for 30 complexed with JAK1, 4E4M for 30 complexed
with JAK2, and 4E4N for 49 complexed with JAK1.
AUTHOR INFORMATION
Corresponding Author
*Tel: +44 1279 645624. Fax: +44 1279 645646. E-mail: janusz.
kulagowski@glpg.
■
Present Addresses
▽Department of Infectious Disease, MedImmune, Gaithersburg,
Maryland, United States.
⧫Cancer Research UK Drug Discovery Unit, Paterson Institute
for Cancer Research, University of Manchester, Wilmslow
Road, Manchester M20 4BX, United Kingdom.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We wish to thank Peter Crackett for the provision of inter-
mediates, Russell Scammel, Michael Podmore and Neil Bayliss for
analytical support, Erlie Delarosa (hepatocyte stability), and Ning
Liu (microsomal stability) for in vitro ADME support, and Peter
Dragovich and David Clark for editorial assistance.
ABBREVIATIONS USED
■
AcOH, Acetic acid; BME, β-mercaptoethanol; CDCl3, deu-
terated chloroform; DMSO-d6, deuterated DMSO; EDCI,
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochlor-
ide; HATU, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetrame-
thyluronium hexafluorophosphate; HOBt, hydroxybenzotria-
zole; LogD7.2, log of partition coefficient between octanol and
pH7.2 aqueous buffer; MCT, methyl cellulose/tween; MDCK,
Madin−Darby canine kidney cells; Papp, apparent permeability;
p-TsOH, para-toluenesulfonic acid; SCX-2, prepacked Isolute
silica-based sorbent with a chemically bonded propylsulfonic
acid functional group; TCEP, tris(2-carboxyethyl)phosphine
(
)-trans-3-(Imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)-
cyclopentyl]-carbamic Acid tert-Butyl Ester (47). A suspension of
72 (300 mg, 0.62 mmol) in methanol (3.2 mL) and THF (3.2 mL)
was treated with 2 M aqueous sodium hydroxide solution (1.6 mL,
3.2 mmol), and the mixture was stirred at ambient temperature for
30 min then at 50 °C for 1 h. The mixture was concentrated under
vacuum and the residue obtained partitioned between DCM and
water. The aqueous phase was extracted with DCM, and the combined
extracts were washed with saturated sodium hydrogen carbonate
solution, and brine, dried (sodium sulfate), and concentrated under
vacuum to afford a yellow glass. This was triturated with ether to give a
solid that was collected, washed with ether, and dried to afford 170 mg
(80%) of 47 as an off-white solid. LCMS (Method 1, ESI): RT = 2.93 min,
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m+H = 342.3.; H NMR (400 MHz, DMSO-d6) δ: 11.83 (s, 1 H),
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NOTE ADDED AFTER ASAP PUBLICATION
■
After this paper was published on the Web June 11, 2012, a
change was made to ref 31. The corrected version was reposted
June 15, 2012.
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